CN110248650A - Method for enhancing cynapse generation and neural process generation - Google Patents

Abstract

Disclosed herein is a kind of methods for enhancing cynapse generation and/or neural process generation, the accumulation or the aggregation that reduce neurodegeneration, and/or reduce Tau albumen in the object with cochlea cynapse disease or vestibular cynapse disease or central nervous system disease or illness; comprising applying a effective amount of 2 to the object for thering is this to need; 4- disulfonyl base α-Phenyl t-butyl Nitrone (2,4-DSPBN) or its pharmaceutically acceptable salt.This method can further include to object and apply N-acetylcystein (NAC).

Description

Methods for enhancing synaptogenesis and neurite outgrowth

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims US Provisional Application No. 62/415,101, filed October 31, 2016, US Provisional Application No. 62/488,740, filed April 22, 2017, US Provisional Application No. 62/510,596, filed May 24, 2017 and priority to US Provisional Application No. 62/550,345, filed August 25, 2017, the entire contents of which are incorporated herein by reference.

background

Chronic diseases (eg, chronic hearing loss, tinnitus, hyperacusis, presbycusis, or balance disorders) are often associated with noise and/or age-related cochlear synaptopathy and vestibular synaptopathy in patients, associated with hair cell loss It doesn't matter. For example, Kujawa et al., J. Neurosci., 29:14077-14085 (2009) and Lin et al., JARO, 12:605-616 (2011) demonstrated that despite mild noise trauma in mouse and guinea pig models There is normal hair cell complement, but noise-induced loss of inner hair cell neurites (eg, synaptopathies) can be extensive. This mechanism may lead to hearing impairment caused by noise impairment or cumulative age-related hearing loss (eg, presbycusis).

Sergeyenko et al., J. Neurosci., 33: 13686-13694 (2013) Demonstrate progression of cochlear synaptic loss from youth to old age and age-related changes in threshold or hair cell count in a mouse model of presbycusis changes can be seen throughout the cochlea long before. In addition, Makary et al., JARO, 12:711-717 (2011) demonstrated that in aged human ears with fully replenished hair cells, the average loss of spiral ganglion cells (SGCs, i.e. cochlear neurons) at age 95 Up to about 30%, suggesting that neurodegeneration can occur before and/or independently of hair cell loss. Furthermore, in a related study in humans, Viana et al., Hear Res., 327:78–88 (2015) provided that cochlear synaptopathies and degeneration of peripheral nerve axons occur independently of hair cell loss and may contribute to Evidence for presbycusis. These types of diffuse synaptic or SGC loss, although may not necessarily affect hearing thresholds, can lead to difficulties with processing and hearing in noisy environments or lead to associated maladaptive sequelae such as tinnitus or hyperacusis. Cochlear synaptopathies and their functional consequences are also described in Schaette et al., J. Neurosci., 31:13452-13457 (2011), Wan et al., Hear Res., 329:1-10 (2015) and Liberman et al., PLoS One, 11(9):e0162726 (2016).

Ear blast injuries are very common in modern military operations due to improvised explosive devices (IEDs) that can cause sensorineural hearing loss and tinnitus. Tinnitus and hearing loss are the most common adverse medical conditions among veterans with disabilities in relational services. Blast exposure can also cause damage to the central auditory system, and sustained oxidative stress is thought to play a fundamental role in this pathophysiological response.

Tauopathies and cochlear neurodegeneration share oxidative stress as a common pathophysiological correlator and potential transmitter of persistent damage. More specifically, several studies have shown that oxidative stress acts as a direct catalyst to induce hyperphosphorylation and aggregation of Tau. This correlation is further supported by work in superoxide dismutase 2-deficient mice, which, under conditions of chronic oxidative stress, exhibit constitutive hyperphosphorylation of Tau as an early postnatal pathological event that can be catalyzed by high doses Antioxidant treatment effectively relieves. Consistent with this mechanistic advantage, targeted therapy of oxidative stress using antioxidants has been shown to ameliorate a wide range of tauopathies.

In addition, Selkoe, Science, 198:789-791 (2002) observed that Alzheimer's disease begins with hippocampal synaptic dysfunction before neuronal degeneration, and synaptic dysfunction is caused by diffusible oligomers of amyloid beta protein Assembly-induced, and at least one mouse line has shown, that a significant reduction in the number of synapses can lead to damage. Sheng et al., Cold SpringHarb Perspect Biol, 4:a005777 (2012) discuss two major themes in the pathogenesis of Alzheimer's disease. First, oligomeric Ab species have strong detrimental effects on synaptic function and structure, especially on the postsynaptic side; second, decreased presenilin function impairs synaptic transmission and promotes neurodegeneration. In addition, Goldstein et al., Sci.Transl.Med., 4(134):doi:10.1126/scitranslmed.3003716 (2012) reported chronic traumatic injury in wild-type C57BL/6 mice 2 weeks after exposure to a single blast Evidence for encephalopathy (CTE), which is similar to CTE neuropathology observed in American football players, and discloses blast-induced persistent hippocampal-dependent learning and memory deficits with impaired axonal conduction and lack of activity-dependent synaptic transmission long-term enhancement correlation. These observations highlight the importance of synaptogenesis and neuritogenesis for the treatment of Alzheimer's disease, CTE and other diseases of the central nervous system.

Therefore, there is a need to enhance cochlear or vestibular synaptopathies in patients with cochlear or vestibular synaptopathies or diseases or disorders of the central nervous system (especially those associated with chronic hearing loss, tinnitus, allergies, presbycusis, or balance disorders). Methods of synaptogenesis and neurite outgrowth in patients with disease). Furthermore, neurodegeneration and Tau protein aggregation (eg, caused by blast exposure) highlight another need for methods to reduce neurodegeneration and Tau protein accumulation or aggregation.

Overview

The present inventors have successfully met the aforementioned medical needs by providing methods and compositions for enhancing synaptogenesis and neuritegenesis in patients suffering from cochlear synaptopathies or vestibular synapses. Accordingly, at least one aspect of the invention described herein pertains to methods for enhancing synaptogenesis and neurite outgrowth in a subject suffering from cochlear synaptopathies or vestibular synaptopathies, including providing said subjects in need thereof An effective amount of 2,4-disulfonyl alpha-phenyl tert-butylnitrone (2,4-DSPBN) or a pharmaceutically acceptable salt thereof is administered. The present disclosure also provides a method of reducing neurodegeneration or Tau protein accumulation in a subject, comprising administering to the subject an effective 2,4-DSPBN or a pharmaceutically acceptable salt thereof.

In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, sublingually, subdermally, intrathecally, by inhalation, or in the ear locally applied to the subject.

In some embodiments, the method further comprises administering one or more selected from the group consisting of N-acetylcysteine (NAC), acetyl-L-carnitine, glutathione monoethyl ester, ebselen Compounds of , D-methionine, carbamathione and Szeto-Schiller peptides and their functional analogs.

In some embodiments, the method further comprises administering NAC.

In some embodiments, the subject has chronic hearing impairment or chronic hearing loss. In some embodiments, the chronic hearing impairment or chronic hearing loss is caused by aging.

In some embodiments, the chronic hearing impairment or chronic hearing loss is caused by exposure to acute or chronic explosions or noise. In some embodiments, 2,4-DSPBN, or a pharmaceutically acceptable salt thereof, is administered to a subject at least one month after exposure to the explosion or noise. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one year after exposure to the explosion or noise.

In some embodiments, the chronic hearing impairment or chronic hearing loss is caused by an infection. In one embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one month after infection. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one year after infection.

In some embodiments, the chronic hearing impairment or chronic hearing loss is caused by exposure to a toxin. In one embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one month after exposure to the toxin. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one year after exposure to the toxin.

In some embodiments, the subject suffers from tinnitus.

In some embodiments, the subject suffers from hyperacusis.

In some embodiments, the subject has presbycusis.

In some embodiments, the subject has a balance disorder. In some embodiments, the subject has Meniere's disease with synaptic loss.

In some embodiments, administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof results in regeneration of cochlear neurites or vestibular neurites in the subject. In some embodiments, the number of active neural connections on inner hair cells is increased. In some embodiments, the number of synapses in tonotopic regions in the organ of Corti is increased.

In some embodiments, the patient does not experience substantial loss of cochlear hair cells or vestibular hair cells.

Another aspect of the invention described herein relates to a method of enhancing synaptogenesis and neuritegenesis in a subject having a disease or disorder of the central nervous system comprising administering to said subject in need thereof an effective amount of 2,4 - Disulfonyl α-phenyl tert-butylnitrone (2,4-DSPBN) or a pharmaceutically acceptable salt thereof.

In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered in a pharmaceutical composition that includes a pharmaceutically acceptable carrier. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject orally, intravenously, subcutaneously, sublingually, subdermally, intrathecally, by inhalation, or topically in the ear . In some embodiments, the method further comprises co-administering NAC to the subject.

In some embodiments, the subject has a central nervous system disease or disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, frontotemporal dementia, Peak Pick's disease, argentophilic granule dementia, corticobasal degeneration, progressive subcortical hypergliosis, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcification, chronic traumatic encephalopathy Dementia, boxer dementia, pure tangling dementia, Down's syndrome, Gerstmann-Straussler-Scheinker disease, Hallewolden-Spaatz disease Hallervorden-Spatz disease, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pickdisease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing Panencephalitis, myotonic dystrophy, noncoronary neuropathy with neurofibrillary tangles, and post-encephalitic parkinsonism.

These and other features, together with their organization and operation, will become apparent from the following detailed description in conjunction with the accompanying drawings.

Description of drawings

Figure 1 shows that HPN-07 elicits neurogenic responses in mouse spiral ganglion (SG) tissue explants in vitro. HPN-07 caused significant neurite outgrowth relative to untreated controls (NC, normal controls) cultured in the same organotypic medium. The number of neurites radiating from each SG explant and the corresponding length of each of these extensions increased in the presence of HPN-07.

Figure 2 shows that HPV-07 enhances neurite outgrowth induced by canonical cochlear neuro-derived growth factor, brain-derived neurotrophic factor (BDNF). Mouse spiral ganglion explants were cultured in serum-free medium with or without BDNF (10 ng/mL) or BDNF (10 ng/mL) + HPN-07 (0.1 μM) for 48 hours, then fixed and treated with β- Tubulin was immunolabeled to visualize neurites.

Figure 3 shows the average neurite length per cell in the PC12 cell line. Combination treatment of HPN-07 and escalating doses of NGF showed that HPN-07 significantly enhanced NGF-mediated neurite elongation (ie, an increase in mean neurite length) in the neuronal PC12 cell line.

Figure 4 shows the percentage of PC12 cells exhibiting significant neurite outgrowth. Combination treatment with HPN-07 and escalating doses of NGF showed that HPN-07 significantly enhanced NGF-induced neurite outgrowth in neuronal PC12 cells, as evidenced by a significant increase in the percentage of neurite-bearing cells following treatment.

Figure 5 shows the reversal of kainic acid (KA)-induced excitotoxic loss of inner hair cell (IHC) ribbon synaptic integrity by HPN-07.

Figure 6 summarizes quantitative assessment of targeted comparisons of the number and spatial distribution of ribbons in IHC from untreated, BDNF-treated or HPN-07-treated cochlear explants following excitotoxic exposure to KA .

Figure 7 shows presynaptic marker (C-terminal binding protein 2, CtBP2) quantification and postsynaptic marker (GluR2/3) quantification. In the image above, rats were repeatedly exposed to three consecutive mine blasts at 14 psi at 1.5 minute intervals, then injected (i.p.) with HPN-07/NAC one hour after the final blast and then daily for the next 48 hours twice. In the lower panel, rats were exposed to a single 8 psi blast in a shock tube, followed by injections (i.p.) of HPN-07/NAC one hour after the final blast, and then twice daily for the next 48 hours.

Figure 8 shows antioxidant treatment prevents cochlear ribbon synapse loss in response to blast-induced trauma. Confocal imaging shows that relative to blank CtBP2- and GluR2/3-immunolabeling along the IHC basolateral membrane in the middle turn (16kHz region) of the OC in age-matched controls (AC), blast-exposed animals (DF) 21 days post-exposure reduce. Animals treated with the combination of HPN-07 and NAC after blast exposure showed no substantial loss of this foci of IHC synapses in this region of the OC (G-1). Presynaptic ribbons were labeled with anti-CtBP2 antibody (red) and postsynaptic densities were labeled with anti-GluR2/3 antibody (green). The merged image in the far right panel shows the overlapping signal intensities (yellow) for each condition of the two synaptic markers. Confocal images are maximal projections of z-stacks of ribbons within 8-10 IHCs in the 16kHz region. Scale bar = 5 μm in I for AI.

Figure 9 shows that acute therapeutic intervention of HPN-07 and NAC reduces Ribbon synaptic loss within the IHC in the 16-32 kHz region of the OC of blast-exposed animals. OC in a cohort of age-matched placebo and untreated and antioxidant-treated blast-exposed animals after 7 days (7D, A and B) or 21 days (21D, C and D) of blast exposure Synaptic marker counts were performed in IHC in the 2, 4, 8, 16, 32, and 48 kHz regions. A synergistic, statistically significant loss of presynaptic and postsynaptic markers was observed in IHC in the 4-48(7D) and 16-48(21D) kHz regions in untreated, blast-exposed animals . Significant therapeutic effects of antioxidants against the opposite blast-induced loss of these synaptic markers were identified in the 8 and 16 kHz regions of the OC, and were observed in the 32 (7D and 21D) and 48 (7D) kHz regions Treatment had a statistically significant trend. No such therapeutic effect was observed in the 48 kHz region 21 days after blast exposure. Statistical significance of the between-group difference in Ribbon synapse loss is indicated by asterisks: *, ** or *** indicate p<0.05, 0.01 or 0.001, respectively, while statistical significance for antioxidant treatment is indicated by ## indicates p<0.01. Error bars represent standard error of the mean (SEM). Numbers in parentheses indicate the total number of OCs assessed in each cohort.

Figure 10 shows that combinatorial antioxidant intervention prevents blast-induced nerve fiber loss in bony SL. Images in A-C are mid-backs from normal controls (A), blast-exposed rats (B) untreated 21 days post-injury, and blast-exposed rats (C) treated with antioxidants 21 days post-blast Examples of NF-200-positively stained nerve fibers in cross-sectional views of SL (A-C). Compared to normal controls (A) and blast-exposed animals (C) treated with HPN-07 and NAC, more was observed in the SL in the cochlea of untreated blast-exposed animals (B) 21 days after blast exposure. Fewer NF-200 positive nerve fibers. NF-200 positive nerve fibers were quantified in the SL of the middle and basal turns of the cochlea from each experimental cohort, and the density of neurites was estimated and statistically analyzed (D). The number of NF-200-positive neurites in the SL of untreated blast-exposed animals was significantly reduced 21 days after blast exposure compared to NC (p<0.05, *). This reduction in NF-200-positive neurites in the SL was not observed in HPN-07/NAC-treated blast-exposed animals 21 days after blast exposure (p<0.01, ##, vs. untreated blast-exposed animals). compared to animals). No significant changes in neurite density were observed at early time points in any cohort (all p>0.05). Scale bar = 10 μm in C for A-C. Numbers in parentheses indicate the total number of animals assessed in each group at each time point. Error bars in D represent SEM.

Figure 11 shows evidence that antioxidant treatment reduces blast-induced neurodegeneration in SG. Images in A-C are normal controls (A), blast-exposed rats (B) untreated at day 21 post blast, and in the basal gyrus of blast-exposed rats treated at day 21 (C) in rats Example of NF-200 staining in SG. Most neurons in the SG stained positively for NF-200 (light positivity), while some neurons were strongly immunoreactive in each cohort (strong positivity, arrows in A-C). Arrows in A-C indicate neurons that were not immunolabeled with NF-200 antibody. The number of NF-200 light or strong positive neurons in SGs was quantified and their relative percentages in each sample were calculated based on comparison with the total number of neurons (D and E). Differences in these percentiles between groups at each time point were then statistically analyzed. A decrease in the percentage of NF-200 light positive neurons was observed in the SG of untreated blast-exposed animals at day 21 post blast exposure compared to NC (p<0.001, ***). A positive, statistically significant treatment effect on NF-200 light positive cell loss was observed at this time point (21D-B vs 21D-B/T, p<0.001, ###, D). An increased percentage of NF-200 strongly positive neurons was observed in the SG of untreated blast-exposed animals on days 7 and 21 post blast exposure compared to NC (all p<0.01, **). A positive, statistically significant treatment effect on the increase in NF-200 strongly positive cells was observed on day 21 post blast exposure (21D-B vs 21D-B/T, p<0.05, #, E). Numbers in parentheses indicate the total number of animals assessed in each group at each time point. Error bars represent SEM in D and E. Scale bar = 20 μm in C for A-C.

Figure 12 shows that therapeutic intervention of HPN-07 and NAC significantly reduced the pathological increase in NF-68 immunolabeling within SGs in response to blasting. Images in A-C are of SGs from NC rats (A), untreated blast-exposed rats (B) on day 7 post-injury, and animals treated with antioxidants on day 7 post-blast exposure (C) Example of NF-68 immunostaining in the bottom fold. Note that while some small neurons were NF-68 positive in all conditions (arrows in A-C), multiple large NF-68 positive neurons were uniquely evident in blast-exposed animals 7 days after injury (arrow in B). The total number of NF-68 positive neurons in each cohort was counted at each sampling interval, and the percentage of NF-68 positive neurons was calculated and statistically analyzed (D). Significant increases in the number of NF-68 positive neurons were observed in the SGs of untreated blast-exposed animals 7 and 21 days after blast exposure (all p<0.001, ***). Significant antioxidant treatment effects were found at both the 7-day and 21-day time points after blast exposure (all p<0.05, #). Scale bar = 20 μm in C for A-C. Error bars in D represent SEM. Numbers in parentheses indicate the total number of animals assessed in each group at each time point.

Figure 13 shows evidence that antioxidant treatment reduces blast-induced neurodegeneration in SG. Maximum diameter of spiral ganglion neurons was measured and statistically analyzed in untreated and HPN-07/NAC-treated animals 21 days after blast exposure. In all three episodes, significantly reduced somatic diameters were observed in the cochlea of untreated blast-exposed animals compared to the placebo (all p<0.001, ***). In HPN-07/NAC-treated blast-exposed animals, mean somatic cell diameters in all three gyri were statistically indistinguishable from blank controls (all p>0.05). Numbers in parentheses indicate the total number of animals evaluated in each group.

Figure 14 shows that antioxidant treatment reduces explosion-induced accumulation of hyperphosphorylated Tau in SGs. Images in A-C are mid-cycle SGs from normal control animals (A), blast-exposed rats (B) untreated 7 days post-blast, and antioxidant-treated rats (C) 7 days post-injury Example of AT8 immunostaining. The number of AT8-positive SGNs (arrows in B and C) was counted, and the percentage of AT8-positive neurons was calculated and used for statistical comparisons between experimental cohorts at each time point (D). Increased AT8 accumulation was observed in SGNs in untreated and treated rats on day 7 after blast exposure (p<0.01 or 0.001, ** or ***). A significant HPN-07/NAC treatment effect was observed at this time point after blast exposure (p<0.05, #). Scale bar = 20 μm in C for A-C. Numbers in parentheses indicate the total number of animals assessed in each group at each time point. Error bars in D represent SEM.

Figure 15 shows that antioxidant treatment reduces blast-induced accumulation of pathological Tau oligomers in SGs. The images in A-C are the middle back of the SG of normal control rats (A), blast-exposed rats (B) untreated 7 days post-blast, and antioxidant-treated rats (C) 7 days post-injury Example of immunostaining of meso-oligomeric Tau (T22). T22-positive neurons were observed in untreated blast-exposed animals (arrows in B) and antioxidant-treated blast-exposed animals (arrows in C). The number of T22-positive neurons in the SG was quantified, and the percentage of T22-positive neurons in each cohort at each time point was calculated and statistically analyzed (D). Greater numbers of T22-positive neurons were observed in the SG of untreated blast-exposed animals at all time points examined (p<0.05 or 0.001, * or ***). Significant treatment effects were observed at the 7-day and 21-day time points after blast exposure (p<0.05 or 0.01, # or ##), but not at the acute, 24-hour time points after blast exposure (p<0.05 or 0.01, # or ##) >0.05). Scale bar = 20 μm in C for A-C. Numbers in parentheses indicate the total number of animals assessed in each group at each time point. Error bars in D represent SEM.

Figure 16 shows that blast exposure results in simultaneous somatic accumulation of oligomeric Tau and NF-68 fragments in SGN. Images are examples of double labeling of T22 and NF-68 in the SG of the mid-gyrus in untreated animals 24 hours (A-D) or 7 days (E-H) after blast exposure. SGN immunopositivity for somatic NF-68 and nuclear and cytoplasmic T22 was uniquely observed in SGs in response to blasts (green, arrows in A, B, D, E, F and H). Some T22-positive neurons without NF-68 labeling were also observed in the SG (red, arrows in A, E, D and H). Nuclei were stained with DAPI (blue). Scale bar = 5 μm in H for A-H.

Figure 17 shows that antioxidant treatment reduces blast-induced somatic accumulation of Tau in the auditory cortex (AC). Images in A-C are Tau- in the deep layers of normal control rats (A), blast-exposed rats (B) untreated 7 days post-blast, and antioxidant-treated rats (C) 7 days post-injury 1 Example of immunostaining. Tau-1 positive neurons were observed in normal controls (arrows in A), untreated blast-exposed animals (arrows in B) and antioxidant-treated blast-exposed animals (arrows in C). The number of Tau-1 positive neurons in the AC was quantified, and the percentage of Tau-1 positive neurons in each cohort at each time point was calculated and statistically analyzed (D). An increase in the number of neurons with Tau-1 positive soma was observed in the AC at all time points post-injury in blast-exposed animals (p<0.05, 0.001 or 0.001, *, ** or ***) . Antioxidant treatment reduced this abnormal immunostaining pattern in blast-exposed rats at sampling intervals of 7 and 21 days, but statistical significance of the positive treatment effect was only concluded in the 21-day cohort (p<0.001 , ###). Numbers in parentheses indicate the total number of animals assessed in each group at each time point. Scale bar = 20 μm in C for A-C. Error bars represent SEM.

Figure 18 shows that HPN-07/NAC treatment reduces blast-induced oxidative stress in SGs. Images are the basal gyrus of the cochlea from blank (A), blast-exposed rats that were not treated 24 hours after blast exposure (B), and rats treated with HPN-07/NAC 24 hours after blast exposure (C) Example of 8-OHdG immunostaining in SG. The number of 8-OHdG-positive neurons in the SG was quantified, and percentiles of results in each cohort were calculated and statistically analyzed (D). An increased number of 8-OHdG positive neurons was observed in the SG of untreated blast-exposed animals (p<0.001, ***). HPN-07/NAC treatment significantly reduced this blast-induced stress response (p<0.01, ***). Numbers in parentheses indicate the total number of animals evaluated in each group. Scale bar in C = 25 μm for A-C. Error bars represent SEM.

Figure 19 shows that antioxidant treatment protects IHC innervation regions from blast-induced neurite loss. Images in A-F are from blank rats (A, D), blast-exposed animals that were not treated 21 days post blast exposure (B, E), and animals treated with HPN-07/NAC 21 days post blast exposure (C , F) Example of NF-200-positively stained neurites (pink, arrows in A-C) in the IHC region of the middle gyrus of the cochlea. The rectangles in A-C indicate the locations from which D-F images were acquired. Observed along the IHC innervation area in the cochlea of untreated blast-exposed animals compared to normal controls (arrows in D) or blast-exposed animals treated with HPN-07 and NAC (arrows in F) Fewer NF-200 positive neurites (arrows in E). Arrows and brackets in A-C indicate IHC and OHC (green), respectively. Nuclei were stained with DAPI (blue). The number of NF-200 positive neurites adjacent to each IHC in the middle and basal gyri of the cochlea for each cohort was counted at each sampling interval and statistically analyzed (G). The number of NF-200-positive neurites per IHC was significantly reduced in untreated blast-exposed animals at 7 and 21 days post blast (7D-B and 21D-B) compared to normal controls (NC) ( ** or ***, p<0.01 or 0.001). Protection against loss of NF-200-positive neurites in IHC innervated areas was observed in blast-exposed animals treated acutely with HPN-07 and NAC 7 and 21 days after blast exposure compared to untreated blast-exposed animals Significance (# or ###, p<0.05 or 0.001). Error bars represent standard error of the mean (SEM). Numbers in parentheses indicate the total number of animals assessed in each group at each time point. Scale bar for C in A-C, F in D-F = 5 μm.

Figure 20 shows that HPN-07/NAC treatment reduces blast-induced hearing loss. The mean ABR thresholds were shifted between 2-16 kHz comparing treated and untreated animals at three time points after blast exposure. At each time point, the mean ABR threshold shift in blast-exposed animals treated with HPN-07/NAC (B/T) was significantly smaller than that in untreated blast-exposed controls (B). The numbers in parentheses indicate the number of ears in each group. **, *** indicate p<0.01 and 0.001 between treated and untreated blast-exposed animals. ABR threshold shift mediated by HPN-07/NAC was reduced at 7 days (7D) or 21 days (21D) after blasting compared to that observed at 24 hours after blasting (24H), ### indicates p<0.001. Error bars represent SEM.

Figure 21 shows HPN-07 and NAC attenuate noise-induced hearing loss. (A) ABR threshold shift after 1 day of noise; (B) ABR threshold shift after 15 days of noise. HPN-07/NAC treatment attenuated noise-induced hearing loss. * and *** represent p<0.05 and 0.001, respectively, for sample significance at any given frequency by two-way ANOVA followed by Bonferroni post-test.

Figure 22 shows that hair cell loss following noise exposure is minimal and occurs only in the basal region of the cochlea.

Figure 23 shows that HPN-07 and NAC reverse noise-induced loss of excitotoxicity at IHC ribbon synapses in vivo. In Sprague Dawley rats, one-shot noise (110 dB, 8-16 kHz, 2 hours) resulted in 30-40% synaptic loss at higher frequencies. HPN-07/NAC treatment reversed the damage. The noise alone group and the noise + HPN-07/NAC group were compared. ** and *** represent p<0.01 and 0.001, respectively. Ribbon synaptic loss is permanent twenty-four hours after injury, so recovery must be regenerative.

Figure 24 shows that HPN-07 enhances BDNF-induced neurite outgrowth in spiral ganglion neuron explants. Spiral ganglion neurons have an inherent, but limited ability to regenerate nerve fibers after injury. Endogenous neurotrophic factor, brain-derived neurotrophic factor (BDNF), can promote recovery. In addition to BDNF, HPN-07 greatly increased the number and extent of nerve fibers. *** indicates p<0.001 compared to NC. ## indicates p<0.01 compared to BDNF (10 ng/ml).

Figure 25 shows that in experiments in which HPN-07 potentiated BDNF-induced neurite outgrowth in spiral ganglion neuron explants, neurite length was not significantly altered by any treatment compared to normal controls (NC).

Figure 26 shows that HPN-07 enhances NT-3 induced neurite outgrowth. Similar to BDNF, HPN-07 also enhanced NT-3-induced neurite outgrowth. *** indicates p<0.001 compared to NC. # and ## represent p<0.1 and p<0.01, respectively, compared to NT-3 (10 ng/ml).

Figure 27 shows that in experiments in which HPN-07 enhanced NT-3-induced neurite outgrowth, neurite length was not significantly altered by any treatment compared to normal controls (NC).

Figure 28 shows that NHPN-1010 treatment (HPN-07 plus NAC) restores hearing function in a pilot study of established hearing loss. A permanent threshold shift is established through wild explosion damage. Treatment was initiated 4 weeks post-injury with HPN-07 plus NAC administered at 300 mg/kg twice daily for 14 days. 14 weeks post-injury (8 weeks post-treatment) showed improvement in ABR thresholds compared to pre-treatment.

Figure 29 shows that NHPN-1010 treatment (HPN-07 plus NAC) restores IHC ribbon synapse number in an established chronic hearing loss model. NHPN-1010 treatment was initiated 4 weeks after injury and administered daily for 14 days. Ribbon synapse counts recovered significantly at 8 weeks post-injury.

Figure 30 shows that NHPN-1010 treatment (HPN-07 plus NAC) resulted in restoration of ABR wave I amplitude in an established chronic hearing loss model. Amplitudes in response to 4 kHz and 8 kHz stimuli at 80 and 70 dB SPL were decreased by blast exposure in the placebo group and continued to decrease over time. Eight weeks after NHPN-1010 treatment, the reduction in amplitude in response to 8 kHz stimulation at 80 dB SPL fully recovered to pre-treatment levels.

Figure 31 shows that Ribbon synapses are reduced after blasting at 16, 32, 48 kHz and maintained/recovered after treatment with NHPN-1010 (HPN-07 plus NAC).

Figure 32 shows that NHPN-1010 treatment (HPN-07 plus NAC) is able to improve blast effects in primary auditory afferent neurons - reducing retrograde neurodegeneration.

Figure 33 shows that HPN-07 and NAC reverse noise-induced synaptic loss in vivo. In this experiment, five doses of HPN-07/NAC treatment appeared to reverse noise-induced synaptic loss more effectively than two doses. Comparisons were made between the noise alone group and the noise + HPN-07/NAC group (5 doses). ** indicates p<0.01.

Figure 34 shows that HPN-07/NAC HPN-07/NAC restores noise-induced ABR amplitude abnormalities in vivo. Compared with the untreated group, the ABR wave I amplitude and wave V/I amplitude ratio in the treated group did not change 15 days after noise exposure compared to before noise. There was no change in wave V amplitude between the two groups. **p<0.01, repeated measures two-way ANOVA with significant main effects in frequency.

Figure 35 shows activity-regulated cytoskeleton-associated protein (ARC) immunostaining in the central auditory system. ARC, also known as Arg3.1, is a plasticity protein. Decreased ARC in the central auditory system is associated with tinnitus. Blast exposure decreased ARC in AC, IC and DCN. HPN-07/NAC treatment normalized ARC expression in AC, IC, and DCN compared to the blast group without treatment. *, *** indicate p<0.05 or 0.001 compared to NC, ### indicates p<0.001 compared to untreated blast group.

Figure 36 shows growth-associated protein 43 (GAP-43) immunostaining and western blotting in the central auditory system. GAP-43 is a membrane-associated phosphoprotein located in axonal growth cones. It is a marker of axonal growth, synaptogenesis and synaptic remodeling. Blast exposure upregulated GAP-43 in AC, IC and DCN. HPN-07/NAC treatment normalized GAP-43 expression in AC, IC and DCN compared to untreated blast group.

Figure 37 shows GABAA receptor alpha1 (GABAARal) immunostaining in the dorsal cochlear nucleus (DCN). GABAA receptors are ionotropic receptors and ligand-gated ion channels. Its endogenous ligand is gamma-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system. Blast exposure upregulated GABAARα1 expression in DCA. HPN-07/NAC treatment normalized the expression of GABAARα1 in DCN compared to the untreated blast group.

Figure 38 shows GABAARα1 (red) and GAD67 (green) co-labeling in DCN. GAD67 is a biomarker of inhibitory neurons, indicating that GABAARα1-positive cells are inhibitory neurons.

Figure 39 shows glutamate receptor 2 (GluR2) immunostaining in DCN. GluR2 is an ionotropic receptor for AMPA, an excitatory neurotransmitter in the central nervous system. Overstimulation of glutamate receptors causes neurodegeneration and neuronal damage through excitotoxicity. Blast exposure upregulated GluR2 expression in DCNs. HPN-07/NAC treatment normalized the expression of GluR2 in DCN compared to the untreated blast group.

Figure 40 shows transient receptor potential cation channel subfamily V member 1 (TRPV1) immunostaining in the spiral ganglion (SG). TRPV1, also known as capsaicin receptor and vanilloid receptor 1, is activated by high temperature, acidic conditions, capsaicin, and irritant compounds. Upregulation of TRPV1 in SG is associated with tinnitus. Blast exposure upregulated TRPV1 in the SG. HPN-07/NAC treatment normalized TRPV1 in SGs compared to the untreated blast group.

Detailed Description

The invention described herein provides a method for promoting or enhancing synaptogenesis and neuritegenesis comprising administering to a subject suffering from cochlear synaptopathies or vestibular synaptopathies an effective amount of 2,4-disulfonyl alpha- Phenyl tert-butylnitrone (2,4-DSPBN, or HPN-07) or a pharmaceutically acceptable salt thereof. Optionally, 2,4-DSPBN is co-administered with NAC.

abbreviation

AVCN, anterior ventral cochlear nucleus; AC, auditory cortex; bTBI, blast-induced traumatic brain injury. CtBP2, C-terminal binding protein 2; DCN, dorsal cochlear nucleus; GluR2/3, glutamate receptor 2/3; HPN-07, 2,4-disulfonyl α-phenyl-tert-butylnitrone; IC , inferior colliculus; IHC, inner hair cell; mTBI, traumatic brain injury from mild blast; NAC, N-acetylcysteine; NF, neurofilament; OC, organ of Corti; OHC, outer hair cell ; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; psi, pounds per square inch; PVCN, posterior ventral cochlear nucleus; SNHL, sensorineural hearing loss; SG, spiral ganglion; SGN, spiral nerve Ganglion neuron; SL, helical blade.

2,4-Disulfonyl α-phenyl tert-butylnitrone (2,4-DSPBN)

2,4-Disulfonyl α-phenyl tert-butylnitrone is also known as 2,4-disulfonyl PBN, 2,4-DSPBN, NXY-059 or HPN-07. It has the following structure:

The acid form of this compound has the following structure:

The acid form can be solid or found in low pH solutions. The ionized salt form of the compound exists at higher pH and can be represented by one of the following structures:

In salt form, X is a pharmaceutically acceptable cation. Most commonly, this cation is a monovalent species, such as sodium, potassium, or ammonium, but it can also be polyvalent alone or in combination with a pharmaceutically acceptable monovalent anion, such as calcium and chloride ions, Anions such as bromide, iodide, hydroxyl, nitrate, sulfonate, acetate, tartrate, oxalate, succinate, pamoate, etc.; magnesium with this anion; zinc with this anion, etc. Of these materials, the free acid and simple sodium, potassium or ammonium salts are most preferred, calcium and magnesium salts are also preferred, but somewhat less preferred. 2,4-DSPBN is described in detail in US Patent No. 5,488,145, which is incorporated herein by reference. Salts of 2,4-DSPBN can also be used to promote or enhance synaptogenesis and neurite outgrowth in a manner similar to the use of 2,4-DSPBN as described herein.

To promote or enhance synaptogenesis and neuritegenesis, 2,4-DSPBN can be administered at doses of, for example, between about 1 mg/kg to about 500 mg/kg body weight, or about 5 mg/kg to about 400 mg/kg body weight Between, or between about 10 mg/kg and about 300 mg/kg body weight, or about 10 mg/kg body weight, or about 20 mg/kg body weight, or about 50 mg/kg body weight, or about 100 mg/kg body weight, or about 150 mg/kg Body weight, or about 200 mg/kg body weight, or about 250 mg/kg body weight, or about 300 mg/kg body weight.

To promote or enhance synaptogenesis and neuritegenesis in a human subject, 2,4-DSPBN can be administered in a daily dose, for example, between about 100 mg and about 20,000 mg, or between about 500 mg and about 10,000 mg, or Between about 1,000 mg and about 5,000 mg, or about 100 mg, or about 200 mg, or about 500 mg, or about 1,000 mg, or about 2,000 mg, or about 3,000 mg, or about 5,000 mg, or about 8,000 mg, or about 10,000 mg.

The subject may be administered one dose per day, or two doses per day, or three doses per day, or four doses per day, or five doses per day.

2,4-DSPBN can be combined with NAC to promote or enhance synaptogenesis and neurite outgrowth. In some embodiments, 2,4-DSPBN and NAC are co-administered as a mixture. In some embodiments, 2,4-DSPBN and NAC are administered sequentially or simultaneously as different dosage forms.

In some embodiments, 2,4-DSPBN and optionally NAC are administered orally. Other delivery methods include, but are not limited to, intravenous, subcutaneous, sublingual, subdermal, intrathecal, or topical in the ear. In addition, the active composition can be administered as a nanoparticle or dendrimer formulation. Nanoparticles can be multifunctional and composed of polymers and paramagnetic iron oxide particles to allow the application of external magnetic forces to aid in drug delivery to desired targets, such as the inner ear or dorsal cochlear nucleus. Additionally, the compositions can be formulated with additives known to those skilled in the art to enhance oral absorption and alter bioavailability kinetics.

Instead of or in addition to 2,4-DSPBN, other nitrone compounds can also be used to promote or enhance synaptogenesis and neurite outgrowth. In some embodiments, the nitrone compound is selected from phenylbutylnitrone (PBN) and derivatives thereof. In some embodiments, the nitrone compound is PBN. In some embodiments, the nitrone compound is 4-hydroxy-α-phenylbutylnitrone (4-OHPBN). In some embodiments, the nitrone compound is 2-sulfonyl-α-phenyl-tert-butylnitrone (S-PBN).

Accordingly, the present application expressly covers the use of any of the above-mentioned nitrone compounds in place of 2,4-DSPBN or in addition to 2,4-DSPBN in all embodiments disclosed herein. Accordingly, methods are disclosed wherein phenylbutylnitrone (PBN), 4-hydroxy-α-phenylbutylnitrone (4-OHPBN) and 2-sulfonyl-α-phenyl tert-butylnitrone One or more of the ketones (S-PBN) were used in place of or in addition to 2,4-DSPBN.

Methods of enhancing synaptogenesis and/or neurite outgrowth

The invention described herein provides a method of promoting or enhancing synaptogenesis and neuritegenesis comprising administering to a subject suffering from cochlear synaptopathies or vestibular synaptopathies an effective amount of 2,4-DSPBN or a pharmaceutically acceptable amount thereof of salt. Optionally, 2,4-DSPBN is co-administered with NAC. Also provided is a method of promoting or enhancing synaptogenesis or neuropathy in a subject with cochlear synaptopathies or vestibular synaptopathies, comprising administering to a subject in need thereof an effective amount of 2,4-disulfonyl α-phenyl tert-butylnitrone or a pharmaceutically acceptable salt thereof.

Synaptopathies refer to disorders of the brain, spinal cord, or peripheral nervous system associated with dysfunction or loss of synapses. Chronic hearing loss, tinnitus, hyperacusis, presbycusis, or balance disorders are often associated with noise- and age-related cochlear or vestibular synaptopathies, not hair cell loss. Cochlear synaptopathies are described in Kujawa et al., J. Neurosci., 29:14077-14085 (2009); Lin et al., JARO, 12:605-616 (2011); Sergeyenko et al., J. Neurosci. , 33:13686-13694 (2013); Makary et al., JARO, 12:711-717 (2011); Viana et al., Hear Res., 327:78–88 (2015); Schaette et al., J. In Neurosci., 31:13452-13457 (2011); Wan et al., Hear Res., 329:1-10 (2015); and Liberman et al., PLoS One, 11(9):e0162726 (2016), All of these are incorporated herein by reference in their entirety.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with chronic hearing impairment or chronic hearing loss, wherein 2,4-DSPBN or The pharmaceutically acceptable salts and optional NAC thereof enhance the regeneration of cochlear neurites to a level sufficient to deliver a therapeutic benefit for chronic hearing impairment or chronic hearing loss to the subject. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to patients with hearing impairment or hearing loss for at least one month, at least three months, at least six months, at least one month years, or at least two years.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient suffering from chronic hearing impairment or chronic hearing loss caused by aging, wherein 2,4, 4-DSPBN, or a pharmaceutically acceptable salt thereof, and optionally NAC, enhances regeneration of cochlear neurites to a level sufficient to deliver a therapeutic benefit to a subject against chronic hearing impairment or chronic hearing loss caused by aging. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a subject at least 65 years old, or at least 70 years old, or at least 75 years old, or at least 80 years old.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient suffering from chronic hearing impairment or chronic hearing loss caused by exposure to explosions or noise , wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC enhances regeneration of cochlear neurites sufficient to deliver to a subject a treatment for chronic hearing impairment or chronic hearing loss caused by exposure to blasts or noise level of benefit.

In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered for at least one month, at least three months, at least six months, at least one year after exposure to explosion or noise or objects of at least two years. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is not within two weeks, within one week, within four days, within two days, within one day, within 12 hours of exposure to the explosion or noise or administered to subjects within 4 hours.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient suffering from chronic hearing impairment or chronic hearing loss caused by infection, wherein 2, 4-DSPBN, or a pharmaceutically acceptable salt thereof, and optionally NAC, enhances regeneration of cochlear neurites to a level sufficient to deliver a therapeutic benefit to a subject for chronic hearing impairment or chronic hearing loss caused by infection.

In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered at least one month, at least three months, at least six months, at least one year, or at least two years after infection Object.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient suffering from chronic hearing impairment or chronic hearing loss caused by exposure to the toxin, wherein 2,4-DSPBN, or a pharmaceutically acceptable salt thereof, and optionally NAC, enhances regeneration of cochlear neurites to a level sufficient to deliver a therapeutic benefit to a subject against chronic hearing impairment or chronic hearing loss caused by exposure to the toxin.

In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered at least one month, at least three months, at least six months, at least one year, or at least one month after exposure to the toxin object for two years.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient suffering from tinnitus, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance the regeneration of cochlear neurites to a level sufficient to deliver therapeutic benefits for tinnitus to the subject. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to patients suffering from tinnitus for at least one month, at least three months, at least six months, at least six months, at least six months Objects of one year or at least two years.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with hyperacusis, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered The salt and optional NAC enhance the regeneration of cochlear neurites to a level sufficient to deliver a therapeutic benefit for hyperacusis to the subject. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to patients with hyperacusis for at least one month, at least three months, at least six months, at least one year, or at least object for two years.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with presbycusis, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a human patient with presbycusis The received salt and optional NAC enhance the regeneration of cochlear neurites to a level sufficient to deliver a therapeutic benefit for presbycusis to the subject. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to patients with presbycusis for at least one month, at least three months, at least six months, at least one year, or Subjects of at least two years.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with a balance disorder, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance the regeneration of cochlear neurites to a level sufficient to deliver therapeutic benefits for balance disorders to the subject. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to patients with a balance disorder for at least one month, at least three months, at least six months, at least one year, or at least object for two years.

In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient suffering from Meniere's disease with synaptic loss, wherein 2,4 - DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC enhances regeneration of cochlear neurites to a level sufficient to deliver therapeutic benefit to the subject for Meniere's disease with synaptic loss. In some embodiments, 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to patients with Meniere's disease with synaptic loss for at least one month, at least three months, at least six months Months, at least one year, or at least two years.

In some embodiments, administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC increases the rate of regeneration of cochlear neurites or vestibular neurites by at least 10%, at least 20%, at least 50%, At least 100%, at least 200%, at least 500% or at least 1000%.

In some embodiments, the administration of 2,4-DSPBN, or a pharmaceutically acceptable salt thereof, and optionally NAC, results in the presence of a The number of active neural connections is increased by at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%. Methods for measuring the number of increased active neural connections on inner hair cells are described in Kujawa et al., J. Neurosci., 29:14077-14085 (2009) and Liberman et al., PLoS One, 11(9) :e0162726 (2016).

In some embodiments, administration of 2,4-DSPBN, or a pharmaceutically acceptable salt thereof, and optionally NAC, results in an increase in the level of The number of synapses (eg, ribbon synapses) in the audio region is increased by at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%. Methods for measuring the number of synapses in audio regions in the organ of Corti are described in Kujawa et al., J. Neurosci., 29:14077-14085 (2009) and Liberman et al., PLoS One, 11(9):e0162726 (2016).

In some embodiments, administration of 2,4-DSPBN, or a pharmaceutically acceptable salt thereof, and optionally NAC causes IHC ribbon synapses to count after one or two or four weeks of treatment, or after more than four weeks of treatment An increase of at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%.

In some embodiments, administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC after one or two or four weeks of treatment, or after more than four weeks of treatment, causes AC, IC and/or The expression of ARC in the DCN is increased by at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%.

In some embodiments, administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC after one or two or four weeks of treatment, or after more than four weeks of treatment, causes AC, IC and/or The expression of GAP-43 in DCN is increased by at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%.

In some embodiments, administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC results in the expression of GABAA Rα1 in DCN after one or two or four weeks of treatment, or after more than four weeks of treatment A reduction of at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%.

In some embodiments, administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC reduces the expression of GluR2 in DCN after one or two or four weeks of treatment, or after more than four weeks of treatment At least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%.

In some embodiments, administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC reduces the expression of TRPV1 in the SG after one or two or four weeks of treatment, or after more than four weeks of treatment At least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%.

Methods of reducing neurodegeneration and/or accumulation of tau protein in the auditory system

Cochlear nerve degeneration is often accompanied by loss of hair cells due to aging, ototoxicity, or exposure to loud noise or explosion overpressure. Nontransgenic rats exposed to explosive overpressure exhibited significant somatic accumulation of neurotoxic variants of the microtubule-associated protein Tau in the hippocampus. In another aspect, the present disclosure relates to reducing neurodegeneration and pathological Tau accumulation in the auditory system in response to blast exposure, and evaluating the potential therapeutic efficacy of antioxidants to short-circuit this pathological process. Blast injury causes Ribbon synaptic loss and retrograde neurodegeneration in the cochlea of untreated animals. In neurons from the peripheral and central auditory systems, concomitant perikaryal accumulation of neurofilament light chains and pathological Tau oligomers was observed spanning from the spiral ganglia to the auditory cortex.

Due to its consistent accumulation pattern and well-documented neurotoxicity, the accumulation of pathological Tau oligomers can actively contribute to blast-induced cochlear nerve degeneration. Therapeutic intervention using a combination regimen of 2,4-disulfonyl α-phenyl-tert-butylnitrone (HPN-07) and N-acetylcysteine (NAC) significantly reduced pathological tau accumulation and cochlear and Signs of ongoing neurodegeneration in the auditory cortex. The present disclosure provides that the combination of HPN-07 and NAC administered shortly after blast exposure can serve as a potential therapeutic strategy for protecting auditory function in military personnel or civilians with blast-induced traumatic brain injury.

Accordingly, the present disclosure described herein provides a method for reducing or slowing neurodegeneration and/or Tau protein accumulation in the auditory system comprising administering to a subject an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof. Optionally, 2,4-DSPBN is co-administered with NAC. In another embodiment, 2,4-DSPBN is co-administered with a Tau aggregation inhibitor. In the present disclosure, the term "Tau" refers to the native monomeric form of Tau, or other conformational isomers of Tau, such as oligomers or aggregates of Tau. The term "Tau" is also used collectively to refer to all types and forms of Tau. Tau proteins function to stabilize microtubules, which are abundant in nerve cells and to a much lesser extent in oligodendrocytes and astrocytes. Pathologies of the nervous system can develop, such as Alzheimer's disease, when Tau proteins become defective and fail to stabilize microtubules sufficiently.

In one embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In another embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, sublingually, subdermally, intrathecally, by inhalation, or topically in the ear object.

In some embodiments, the method further comprises administering one or more selected from the group consisting of N-acetylcysteine, acetyl-L-carnitine, glutathione monoethyl ester, ebselen, D- Compounds of methionine, carbamathione and Szeto-Schiller peptides and their functional analogs. In other embodiments, the method further comprises administering N-acetylcysteine.

In one embodiment, neurodegeneration is caused by aging, or exposure to acute or chronic blasts or noise. In another embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one month after exposure to the explosion or noise. In one embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject for at least one year after exposure to an explosion or noise.

In one embodiment, the neurodegeneration is caused by infection. In one embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one month after infection. In another embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one year after infection.

In one embodiment, neurodegeneration is caused by exposure to a toxin. In another embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one month after exposure to the toxin. In one embodiment, 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a subject at least one year after exposure to the toxin.

In one embodiment, wherein administration of 2,4-DSPBN or a pharmaceutically acceptable salt thereof reduces the accumulation of Tau protein in the subject. In one embodiment, Tau protein accumulates in the auditory system. In some embodiments, the method further comprises administering a Tau aggregation inhibitor. Tau aggregation inhibitors can be covalent or non-covalent inhibitors. Non-limiting examples of tau aggregation inhibitors include curcumin, molecular tweezers (eg CLR01), phthalocyanine tetrasulfonate, oleocanthal, cinnamaldehyde, baicalein, isoproterenol, Dopamine, dobutamine, levodopa, levodopa/carbidopa, trimetoquinol, hexoprenaline, methyldopa, and droxidopa.

Methods of treating diseases of the central nervous system

The invention described herein also provides a method of promoting or enhancing synaptogenesis and neurite outgrowth in the central nervous system of a subject in need thereof, comprising administering to the subject an effective amount of 2,4-DSPBN or a pharmaceutically acceptable amount thereof. An acceptable salt, wherein the subject suffers from a central nervous system disease or disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, frontotemporal dementia, Pick's disease, Silver particle dementia, corticobasal degeneration, progressive subcortical hypergliosis, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcifications, chronic traumatic encephalopathy, boxer dementia, pure tangles Dementia, Down syndrome, Gerstmann-Strausner syndrome, Hallewolden-Spatz disease, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prions Protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, noncoronary neuropathy with neurofibrillary tangles, and post-encephalitic parkinsonism. Optionally, 2,4-DSPBN is co-administered with an effective amount of NAC. 2,4-DSPBN and NAC can be co-administered simultaneously or sequentially. 2,4-DSPBN and NAC can be co-administered in one composition or in separate compositions.

Another aspect of the invention relates to methods of treating Alzheimer's disease. In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with Alzheimer's disease, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a human patient with Alzheimer's disease The acceptable salts above and optionally NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological Tau protein in the central nervous system sufficient to deliver a treatment for Alzheimer's disease to the patient level of benefit.

Another aspect of the present invention relates to methods of treating Parkinson's disease. In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with Parkinson's disease, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a human patient with Parkinson's disease Acceptable salts and optionally NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological Tau protein in the central nervous system sufficient to deliver therapeutic benefits for Parkinson's disease to the patient Level. In some embodiments, the method reduces the accumulation of Tau protein in the central nervous system of the patient.

Another aspect of the invention relates to a method of treating progressive supranuclear palsy (PSP). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with PSP, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for PSP to the patient.

Another aspect of the invention relates to methods of treating frontotemporal dementia (FTD). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with FTD, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver therapeutic benefit for FTD to the patient.

Another aspect of the invention relates to a method of treating Pick's disease. In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with Pick's disease, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered The received salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for Pick's disease to the patient.

Another aspect of the present invention relates to a method of treating arginophilic granular dementia (AGD). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with AGD, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for AGD to the patient.

Another aspect of the invention relates to methods of treating corticobasal degeneration (CBD). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with CBD, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver therapeutic benefit to CBD to the patient.

Another aspect of the invention relates to methods of treating progressive subcortical hypergliosis (PSG). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with PSG, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver therapeutic benefit to PSG to the patient.

Another aspect of the invention relates to methods of treating amyotrophic lateral sclerosis (ALS). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with ALS, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for ALS to the patient.

Another aspect of the invention relates to a method of treating diffuse neurofibrillary tangles with calcification (DNTC). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with DNTC, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit to DNTC to the patient.

Another aspect of the invention relates to methods of treating chronic traumatic encephalopathy. In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with chronic traumatic encephalopathy, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a human patient with chronic traumatic encephalopathy The acceptable salts above and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological Tau protein in the central nervous system sufficient to deliver treatment to patients with chronic traumatic encephalopathy level of benefit.

Another aspect of the present invention relates to a method of treating boxer dementia (DP). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with DP, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit against DP to the patient.

Another aspect of the present invention relates to a method of treating pure tangling dementia (TOD). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with TOD, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for TOD to the patient.

Another aspect of the invention relates to a method of treating Down's syndrome. In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with Down syndrome, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a human patient with Down syndrome Acceptable salts and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system sufficient to deliver therapeutic benefits for Down syndrome to patients Level.

Another aspect of the invention relates to methods of treating Gerstmann-Straussner Syndrome (GSS). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with GSS, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver therapeutic benefit against GSS to the patient.

Another aspect of the present invention relates to a method of treating Hallewolden-Spatz disease (HSD). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with HSD, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for HSD to the patient.

Another aspect of the invention relates to methods of treating Creutzfeldt-Jakob disease (CJD). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with CJD, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver therapeutic benefit for CJD to the patient.

Another aspect of the invention relates to methods of treating multiple system atrophy (MSA). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with MSA, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for MSA to the patient.

Another aspect of the invention relates to a method of treating Niemann-Pick disease type C (NPC). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with NPC, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit against NPC to the patient.

Another aspect of the invention relates to a method of treating prion protein cerebral amyloid angiopathy (PrP-CAA). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with PrP-CAA, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered The received salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological Tau protein in the central nervous system to a level sufficient to deliver therapeutic benefit to PrP-CAA to the patient.

Another aspect of the invention relates to a method of treating subacute sclerosing panencephalitis (SSPE). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with SSPE, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neuritegenesis and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver a therapeutic benefit for SSPE to the patient.

Another aspect of the invention relates to a method of treating myotonic dystrophy. In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with myotonic dystrophy, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to a human patient with myotonic dystrophy The acceptable salts above and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological Tau protein in the central nervous system sufficient to deliver to the patient a treatment for myotonic dystrophy level of benefit.

Another aspect of the invention relates to a method of treating non-coronary neuropathy with neurofibrillary tangles. In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient having non-coronary neuropathy with neurofibrillary tangles, wherein 2,4 - DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC enhances neuritegenesis and/or synaptogenesis, and/or reduces the amount of pathological Tau protein in the central nervous system sufficient to deliver to the patient Levels of therapeutic benefit in neurofibrillary tangles in noncoronary neuropathy.

Another aspect of the invention relates to methods of treating post-encephalitic Parkinson's syndrome (PEP). In some embodiments, an effective amount of 2,4-DSPBN or a pharmaceutically acceptable salt thereof and optionally NAC is administered to a human patient with PEP, wherein 2,4-DSPBN or a pharmaceutically acceptable salt thereof The salt and optional NAC enhance neurite outgrowth and/or synaptogenesis, and/or reduce the amount of pathological tau protein in the central nervous system to a level sufficient to deliver therapeutic benefit to the patient against PEP.

In some embodiments, the method reduces accumulation of Tau protein in the central nervous system of a patient with a central nervous system disease or disorder selected from Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy , frontotemporal dementia, Pick's disease, argentophilic granule dementia, corticobasal degeneration, progressive subcortical hypergliosis, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcification, chronic Traumatic encephalopathy, boxer dementia, pure tangling dementia, Down syndrome, Gerstmann-Straussner syndrome, Hallewolden-Spatz disease, Creutzfeldt-Jakob disease, multiple system atrophy , Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, noncoronary neuropathy with neurofibrillary tangles, and post-encephalitic Parkinson's disease syndrome.

In some embodiments, the method slows, stops or reverses neurodegeneration in the central nervous system of a patient with a central nervous system disease or disorder selected from Alzheimer's disease, Parkinson's disease, progressive nuclear disease Superior paralysis, frontotemporal dementia, Pick's disease, argentophilic granule dementia, corticobasal degeneration, progressive subcortical hypergliosis, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcifications disease, chronic traumatic encephalopathy, boxer dementia, pure tangling dementia, Down syndrome, Gerstmann-Straussner syndrome, Hallewolden-Spaatz disease, Creutzfeldt-Jakob disease, Multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, noncoronary neuropathy with neurofibrillary tangles, and post-encephalitis Parkinson's syndrome.

Other implementations

Embodiment 1. A method of enhancing synaptogenesis and neuritegenesis in a subject suffering from cochlear synaptopathies or vestibular synaptopathies, comprising administering to said subject in need thereof an effective amount of 2,4-disulfonyl alpha - Phenyl tert-butylnitrone (2,4-DSPBN) or a pharmaceutically acceptable salt thereof.

Embodiment 2. The method according to embodiment 1, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

Embodiment 3. The method of embodiment 1, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, sublingually, subcutaneously, intrathecally, by inhalation, or in the ear locally administered to the subject.

Embodiment 4. The method of embodiment 1, wherein the method further comprises administering one or more selected from the group consisting of N-acetylcysteine, acetyl-L-carnitine, glutathione monoethyl ester, ebselen Compounds of morpholino, D-methionine, carbamathione and Szeto-Schiller peptides and their functional analogs.

Embodiment 5. The method of embodiment 1, wherein the method further comprises administering N-acetylcysteine.

Embodiment 6. The method of embodiment 1, wherein the subject has chronic hearing impairment or chronic hearing loss.

Embodiment 7. The method of embodiment 6, wherein the chronic hearing impairment or chronic hearing loss is caused by aging.

Embodiment 8. The method of embodiment 6, wherein the chronic hearing impairment or chronic hearing loss is caused by exposure to acute or chronic explosions or noise.

Embodiment 9. The method of embodiment 8, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after exposure to the explosion or noise.

Embodiment 10. The method of embodiment 8, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after exposure to the explosion or noise.

Embodiment 11. The method of embodiment 6, wherein the chronic hearing impairment or chronic hearing loss is caused by an infection.

Embodiment 12. The method of embodiment 11, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after infection.

Embodiment 13. The method of embodiment 11, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after infection.

Embodiment 14. The method of embodiment 6, wherein the chronic hearing impairment or chronic hearing loss is caused by exposure to a toxin.

Embodiment 15. The method of embodiment 14, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after exposure to the toxin.

Embodiment 16. The method of embodiment 14, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after exposure to the toxin.

Embodiment 17. The method of embodiment 1, wherein the subject also suffers from tinnitus.

Embodiment 18. The method of embodiment 1, wherein the subject also suffers from hyperacusis.

Embodiment 19. The method of embodiment 1, wherein the subject also suffers from presbycusis.

Embodiment 20. The method of embodiment 1, wherein the subject also suffers from a balance disorder or Meniere's disease with synaptic loss.

Embodiment 21. The method of embodiment 1, wherein administration of the 2,4-DSPBN or a pharmaceutically acceptable salt thereof enhances regeneration of cochlear or vestibular neurites in the subject.

Embodiment 22. The method of embodiment 1, wherein the number of active neural connections on inner hair cells is increased in the subject.

Embodiment 23. The method of embodiment 1, wherein the number of synapses in the audio region in the organ of Corti is increased in the subject.

Embodiment 24. The method of embodiment 1, wherein the subject does not experience substantial loss of cochlear hair cells or vestibular hair cells.

Embodiment 25. The method of embodiment 1, wherein the subject has experienced substantial loss of cochlear hair cells or vestibular hair cells.

Embodiment 26. A method of enhancing synaptogenesis or neuritegenesis in a subject suffering from cochlear synaptopathies or vestibular synaptopathies, comprising administering to said subject in need thereof an effective amount of 2,4-disulfonyl alpha - Phenyl tert-butylnitrone (2,4-DSPBN) or a pharmaceutically acceptable salt thereof.

Embodiment 27. A method for reducing neurodegeneration in a subject, comprising administering to said subject in need thereof an effective amount of 2,4-disulfonyl alpha-phenyl tert-butylnitrone (2,4-DSPBN) or its Pharmaceutically acceptable salts.

Embodiment 28. The method according to embodiment 27, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

Embodiment 29. The method of embodiment 27, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, sublingually, subcutaneously, intrathecally, by inhalation, or in the ear locally administered to the subject.

Embodiment 30. The method of embodiment 27, wherein the method further comprises administering one or more selected from the group consisting of N-acetylcysteine, acetyl-L-carnitine, glutathione monoethyl ester, ebselen Compounds of morpholino, D-methionine, carbamathione and Szeto-Schiller peptides and their functional analogs.

Embodiment 31. The method of embodiment 27, wherein the method further comprises administering N-acetylcysteine.

Embodiment 32. The method of embodiment 27, wherein the neurodegeneration is caused by aging.

Embodiment 33. The method of embodiment 27, wherein the neurodegeneration is caused by exposure to an explosion or noise, acute or chronic.

Embodiment 34. The method of embodiment 33, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after exposure to the explosion or noise.

Embodiment 35. The method of embodiment 33, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after exposure to the explosion or noise.

Embodiment 36. The method of embodiment 27, wherein the neurodegeneration is caused by an infection.

Embodiment 37. The method of embodiment 36, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after infection.

Embodiment 38. The method of embodiment 36, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after infection.

Embodiment 39. The method of embodiment 27, wherein the neurodegeneration is caused by exposure to a toxin.

Embodiment 40. The method of embodiment 39, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after exposure to the toxin.

Embodiment 41. The method of embodiment 39, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after exposure to the toxin.

Embodiment 42. The method of embodiment 27, wherein the administration of the 2,4-DSPBN or a pharmaceutically acceptable salt thereof reduces the accumulation of tau protein in the subject.

Embodiment 43. The method of embodiment 42, wherein the Tau protein accumulates in the auditory system.

Embodiment 44. The method of embodiment 27, further comprising administering a tau aggregate inhibitor.

Embodiment 45. A method for reducing the accumulation of Tau protein in a subject comprising administering to said subject in need thereof an effective amount of 2,4-disulfonyl alpha-phenyl tert-butylnitrone (2,4-DSPBN) or a pharmaceutically acceptable salt thereof.

Embodiment 46. The method according to embodiment 45, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

Embodiment 47. The method of embodiment 45, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, sublingually, subcutaneously, intrathecally, by inhalation, or in the ear locally administered to the subject.

Embodiment 48. The method of embodiment 45, wherein the method further comprises administering one or more selected from the group consisting of N-acetylcysteine, acetyl-L-carnitine, glutathione monoethyl ester, ebselen Compounds of morpholino, D-methionine, carbamathione and Szeto-Schiller peptides and their functional analogs.

Embodiment 49. The method of embodiment 45, wherein the method further comprises administering N-acetylcysteine.

Embodiment 50. The method of embodiment 45, wherein the accumulation of Tau protein is caused by aging.

Embodiment 51. The method of embodiment 45, wherein the accumulation of tau protein is caused by exposure to acute or chronic blast or noise.

Embodiment 52. The method of embodiment 51, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after exposure to the explosion or noise.

Embodiment 53. The method of embodiment 51, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after exposure to the explosion or noise.

Embodiment 54. The method of embodiment 45, wherein the accumulation of Tau protein is caused by infection.

Embodiment 55. The method of embodiment 54, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after infection.

Embodiment 56. The method of embodiment 54, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after infection.

Embodiment 57. The method of embodiment 45, wherein the accumulation of Tau protein is caused by exposure to a toxin.

Embodiment 58. The method of embodiment 57, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one month after exposure to the toxin.

Embodiment 59. The method of embodiment 57, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered to the subject at least one year after exposure to the toxin.

Embodiment 60. The method of embodiment 45, further comprising administering a tau aggregate inhibitor.

Embodiment 61. A method of enhancing synaptogenesis and neuritegenesis in a subject suffering from a disease or disorder of the central nervous system, comprising administering to said subject in need thereof an effective amount of 2,4-disulfonyl alpha-phenyl tert-Butylnitrone (2,4-DSPBN) or a pharmaceutically acceptable salt thereof.

Embodiment 62. The method of embodiment 61, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

Embodiment 63. The method of embodiment 61, wherein the 2,4-DSPBN or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, sublingually, subcutaneously, intrathecally, by inhalation, or in the ear locally administered to the subject.

Embodiment 64. The method of embodiment 61, wherein the method further comprises administering to the subject N-acetylcysteine.

Embodiment 65. The method of embodiment 61, wherein the subject has Alzheimer's disease.

Embodiment 66. The method of embodiment 61, wherein the subject has Parkinson's disease.

Embodiment 67. The method of embodiment 61, wherein the subject has progressive supranuclear palsy.

Embodiment 68. The method of embodiment 61, wherein the subject has frontotemporal dementia.

Embodiment 69. The method of embodiment 61, wherein the subject has Pick's disease.

Embodiment 70. The method according to embodiment 61, wherein the subject has argentophilic dementia.

Embodiment 71. The method of embodiment 61, wherein the subject has corticobasal degeneration.

Embodiment 72. The method of embodiment 61, wherein the subject has progressive subcortical hypergliosis.

Embodiment 73. The method of embodiment 61, wherein the subject has amyotrophic lateral sclerosis.

Embodiment 74. The method of embodiment 61, wherein the subject has diffuse neurofibrillary tangles with calcification.

Embodiment 75. The method of embodiment 61, wherein the subject has boxer dementia.

Embodiment 76. The method of embodiment 61, wherein the subject has pure tangling dementia.

Embodiment 77. The method of embodiment 61, wherein the subject has Down syndrome.

Embodiment 78. The method of embodiment 61, wherein the subject has Gerstmann-Strausner syndrome.

Embodiment 79. The method according to embodiment 61, wherein the subject has Hallerwarden-Spatz disease.

Embodiment 80. The method of embodiment 61, wherein the subject has Creutzfeldt-Jakob disease.

Embodiment 81. The method of embodiment 61, wherein the subject has multiple system atrophy.

Embodiment 82. The method of embodiment 61, wherein the subject has Niemann-Pick type C disease.

Embodiment 83. The method of embodiment 61, wherein the subject has prion protein cerebral amyloid angiopathy.

Embodiment 84. The method of embodiment 61, wherein the subject has subacute sclerosing panencephalitis.

Embodiment 85. The method of embodiment 61, wherein the subject has myotonic dystrophy.

Embodiment 86. The method of embodiment 61, wherein the subject has non-coronary neuropathy with neurofibrillary tangles.

Embodiment 87. The method of embodiment 61, wherein the subject has post-encephalitic Parkinson's syndrome.

Embodiment 88. The method of embodiment 61, wherein the subject has chronic traumatic encephalopathy.

specific embodiment

The following examples are for illustrative purposes only and should not be construed as limitations on the claimed invention. Those of ordinary skill in the art may use various alternative techniques and procedures that will likewise successfully accomplish the contemplated invention.

Example 1

In a live animal study, noise-injured chinchillas treated with 2,4-disulfonyl α-phenyl-tert-butylnitrone (HPN-07) were found to exhibit significantly more A large population of inner hair cell neurites, and long after the treatment period, these animals uniquely exhibited a progressive degree of functional recovery, suggesting sustained reinnervation of hair cells by HPN-07. This leads to the possibility that HPN-07 has pro-neurogenic properties, especially in cochlear spiral ganglion neurons (SGN). HPN-07 has been tested in three in vitro neurogenic models (cochlear spiral ganglion explants, PC12 cells, and co-cultures of SGN explants with attached hair cells [SGN-HC]). Experimental data from these analyses demonstrate that: (1) HPN-07 promotes neurite outgrowth in spiral ganglion explants without hair cells; (2) HPN-07 enhances nerve growth factor (NGF) induction in PC12 cell line and (3) HPN-07 reversed excitoxic ribbon synaptic loss in SGN-HC co-cultures following excitoxic trauma by kainic acid (KA) , and increased neurite density along the base of the IHC. HPN-07/NAC treatment of live blast-exposed rats induced a significant increase in the number of active neural connections on inner hair cells after blasting and, in some audio regions, resulted in more synapses than normally found in blank, The number of synapses observed in the undamaged ear.

As shown in Figure 1, HPN-07 induced neurogenic responses in mouse spiral ganglion tissue explants in vitro. HPN-07 caused significant neurite outgrowth relative to untreated controls (NC, normal controls) cultured in the same organotypic medium. The number of neurites radiating from each SGN explant and the corresponding length of each of these extensions increased in the presence of HPN-07. In contrast to the need for PBN at a concentration of 10 mM to induce neurite outgrowth in PC12 cells, HPN-07 at a concentration of 100 μM was sufficient to induce neurogenic responses in organotypic cultures of mouse spiral ganglion neurons.

As shown in Figure 2, HPN-07 enhanced neurite outgrowth induced by the classical cochlear neuro-derived growth factor, brain-derived neurotrophic factor (BDNF). Mouse spiral ganglion neuron explants were cultured in serum-free medium with or without BDNF (10 ng/mL) or BDNF (10 ng/mL) + HPN-07 (0.1 μM) for 48 hours, then fixed and treated with β-Tubulin antibody immunolabeling to visualize neurites.

As shown in Figure 3, HPN-07 also enhanced NGF-induced neurite elongation in PC12 cell line. Nerve growth factor (NGF) treatment induced neurite elongation in a dose-dependent manner in PC12 cells, a standard neuronal model for neurite outgrowth studies. In this context, the neurogenic response elicited by this NGF is apparently saturable at concentrations above 10 ng/mL. However, while treatment with HPN-07 alone at low concentrations in this system did not increase neurite length, combination treatment with 1 μM HPN-07 and escalating doses of NGF (1, 5, or 10 ng/mL) showed that HPN- 07 enhanced (at 1 and 5 ng/mL NGF) NGF, and possibly even synergized with NGF (at 10 ng/mL NGF) to promote neurite out. At concentrations of 10 ng/mL NGF and 1 μM HPN-07, mean neurite length significantly exceeded the maximum length achieved in the saturable NGF dose-escalating series (6 days of cell treatment).

Figure 4 depicts the percentage of PC12 cells with significant neurite outgrowth. The pattern observed here is similar to that observed for the treatment-induced increase in mean neurite length. Together, these assessments support the explanation that HPN-07 cooperates with NGF to promote nascent neurite outgrowth, and changes in neurite stability or assembly dynamics to support an increase in mean neurite length, both of which Both are desirable features of IHC reinnervation. Since growth factors are constitutively expressed in the mammalian cochlea and are frequently up-regulated after auditory trauma, this therapeutic property may have a significant clinical impact on restoring auditory function.

Furthermore, as shown in Figure 5, HPN-07 reversed kainic acid (KA)-induced excitotoxic loss of IHC ribbon synaptic integrity. Previous findings were applied to a mouse SGN-IHC co-culture model of excitotoxic trauma. Most presynaptic C-terminal binding protein 2 (CtBP2)-immunoreactive puncta in IHC of uninjured co-cultures of auditory sensory epithelium (organ of Corti) and associated spiral ganglion neurons (NCs) (small red dots) are located at the location of "typical" cellular structures, along the basal surface of the cell, below or around the IHC nuclei (large red spheres). After exposure to the excitotoxic glutamate analog kainic acid (KA), a marked shift from this typical CtBP2 basal immunoreactivity pattern to a more apical distribution pattern occurred. This abnormal trend correlates with the total loss of co-immunolabeling of the postsynaptic density marker PSD-95, suggesting that apical displacement of CtBP2-labeled puncta may be caused by KA-induced loss of Ribbon synaptic integrity and will have Active synapses are anchored to the basal plane by dissociation of the PSD. Excitotoxic post-treatment with HPN-07 at a concentration (10 μM) shown to cause neurite outgrowth in SG explants reversed this pathological transition and in a manner similar to that achieved with the classical neurotrophic growth factor BDNF. CtBP2 co-labeled basal PSD repopulation IHC. The ability of HPN-07 to restore the structural integrity of ribbon synapses in IHC following excitotoxic trauma in this co-culture model, in contrast to HPN- used to promote synaptogenesis (ie, reinnervation) in this context- 07 Consistent.

Figure 6 summarizes quantitative assessments of targeted comparisons of the number and spatial distribution of forge bands in IHC from untreated, BDNF-treated or HPN-07-treated explants following excitotoxic exposure to KA. Following KA exposure, untreated cultures exhibited a shift in presynaptic density from "typical" (subnuclear) to "apical" (supernuclear) distribution. Both BDNF and HPN-07 reversed this pathological change, underscoring their utility in reinvigorating IHC in this setting. However, HPN-07 treatment resulted in a greater number of total ribbons, closer to that observed in undamaged tissue. Each data point represents a combination of approximately ten independent measurements.

As shown in Figure 7, HPN-07/NAC treatment in live blast-exposed rats induced a significant increase in the number of active neural connections on inner hair cells after blasting and, in some audio regions, tended to exceed the usual Number of synapses observed in blank, uninjured ears. In the image above, rats were repeatedly exposed to three consecutive mine blasts at 14 psi at 1.5 min intervals. Animals were then injected (i.p.) with HPN-07/NAC one hour after the final blast and then twice daily for the next 48 hours. Animals were euthanized three weeks later, and cochlear tissue was fixed, harvested, microsectioned, and treated with targeted ribbon synapses (CtBP2) within inner hair cells and postsynaptic density at the synaptic interface of its associated neurites (GluR2/3) antibodies were co-immunolabeled. The presence of both markers indicates active synaptic connections. A significant therapeutic effect on synapse density was observed in the 16 kHz audio region of the organ of Corti, where there was a trend for a supernumerary number of synapses (relative to blank controls). In the image below, rats were exposed to a single 8 psi blast inside a shock tube. Animals were then injected (i.p.) with HPN-07/NAC one hour after the final blast and then twice daily for the next 48 hours. Animals were euthanized after 8 weeks and cochlear tissue was fixed, harvested, microsectioned, and co-immunolabeled with antibodies targeting CtBP2 and GluR2/3 as described above. A significant treatment effect was observed in the 32 kHz audio region of the organ of Corti in these rats, with a trend for supernumerary synapse numbers (relative to blank controls) in the 8 and 16 kHz regions.

Example 2

Animals, Explosion Exposure, and Drug Administration

In addition to cochlear tissue samples for ribbon synapse assessment, tissue samples used in this study were collected as previously described (Ewert et al., Hear. Res., 285, 29–39 (2012)). Blast exposure, administration of antioxidants, and measurements of auditory brainstem responses were described in detail in our previous report (Ewert et al., Hear. Res., 285, 29–39 (2012)). All procedures regarding the use and handling of animals were reviewed and approved by the Oklahoma Medical Research Foundation (OMRF) Institutional Animal Care and Use Committee and the U.S. Office of Naval Research.

Male Long-Evans colored rats (Harlan Laboratories, Indianapolis, Indiana) weighing between 360 and 400 g were used in this study. Animals were housed in OMRF's animal facility. Each rat was exposed to three consecutive blasts of 14 pounds per square inch (psi) repeated at 1.5 minute intervals. The blast regimen caused significant hearing loss and a low incidence of tympanic membrane rupture (Ewert et al., Hear. Res., 285, 29–39 (2012)). Ears with ruptured tympanic membranes were excluded from the study. Auditory brainstem response (ABR) thresholds and distortion product of otoacoustic emissions (DPOAE) levels were obtained before blast exposure and at sampling intervals of 24 hours (24H), 7 days (7D) and 21 days (21D) after exposure, and reported in our previous publication. Du et al., PLoS One, e80138 (2013); Ewert et al., Hear. Res., 285, 29–39 (2012).

20% NAC solution was purchased from Hospira, Inc. (Lake Forest, IL), and HPN-07 was synthesized and supplied by APAC Pharmaceuticals, LLC (Columbia, MD). Animals in the blast-exposed antioxidant treatment group (B/T) were injected intraperitoneally (i.p.) with a combination of 300 mg/kg NAC plus 300 mg/kg HPN-07 in saline solution (5 mL/kg). Drug administration began 1 hour after blast exposure and continued twice a day for the next two days. Animals in the untreated, blast-exposed group (B) were injected intraperitoneally with an equal volume of normal saline according to the same protocol as the treatment group. An additional 11 rats that were not exposed to the blast or received no drug treatment were used as normal controls (NC).

Collection of Cochlear Tissue for Ribbon Synapse Assessment

One or three weeks after blast exposure, animals were decapitated under deep anesthesia with ketamine and xylazine. The cochlea was rapidly dissected from the temporal bone and placed in cold PBS. The round and oval windows were opened and the bones of the apical turn were removed. A 4% formaldehyde solution in PBS was perfused into the cochlea for tissue fixation. The cochlea was placed in the same fixative for an additional 10 min at 4 °C. After fixation, the cochlea was further dissected in PBS, then blocked for 1 hr in PBS containing 1% Triton X-100 and 5% normal horse serum, followed by rabbit anti-GluR2/3 antibody (Millipore Bioscience, catalog #AB1506, 1: 100) and a combination of mouse anti-C-terminal collectin antibody (CtBP2, BD Transduction Laboratories, catalog #612044, 1:200) for immunolabeling at 37°C for 20 hours. Tissues were rinsed with PBS and incubated with Alexa Fluor488 chicken anti-rabbit (1:1000, Life Technologies, Co., Grand Island, NY) for 1 hour at 37°C. Tissues were washed with PBS and mixed with Alexa Fluor488 goat anti-chicken antibody (1:1000, Life Technologies, Co., Grand Island, NY) and Alexa Fluor568 goat anti-mouse antibody (1:1000, Life Technologies, Co., Grand Island) , NY) at 37°C for 1 hour (Furman et al., 2013). Tissues were counterstained with DAPI (4',6-diamidino-2-phenylindole) for 10 minutes at room temperature to label nuclei and then mounted on slides with antifade medium.

The entire cochlea was photographed with an epi-fluorescence microscope. Cochlear lengths were measured and frequencies were calculated using a custom plug-in ImageJ software (http://www.masseyeandear.org/research/ent/eaton-peabody/epl-histologyresources/). Six frequency positions of the cochlea, 2, 4, 8, 16, 32, 48 kHz, were selected as confocal z-stacks for image acquisition. Images were acquired using a Zeiss LSM-710 confocal microscope (Carl Zeiss Microimaging, LLC, NY) with 1024×1024 pixel frames in the z-plane with 0.5 μm steps. Viewed from the endolymphatic surface of the Organ of Corti, each stack contains six to nine IHCs with a full set of ribbon synapses. Synapses. 3-D morphometric measurements were processed by using Amira 3D software (FEI, Burlington, MA). All quantitative analyses were performed using raw image stacking. Presynaptic ribbons (red channel) and postsynaptic densities (green channel) were identified by segmentation, quantified and tracked in the z dimension to avoid stack ambiguity or overestimation in each stack, and according to previously published methods Divide by the total number of IHCs in the microscopic field (Kujawa and Liberman, J. Neurosci., 29, 14077-14085 (2009)).

Collection and Sectioning of Cochlear and Brain Tissue

Animals for cochlear and brain slices in each experimental group (6-8 rats/time point) were euthanized and intracardially perfused with saline 24 hours, 7 days, or 21 days after blasting, followed by 0.1 M phosphoric acid 4% paraformaldehyde in salt-buffered saline (PBS, pH 7.2) was perfused. After removal of the cochlea, brain and brain stem, they were fixed in the same fixative (overnight for cochlea and one week for brain tissue), washed in PBS, and stored in PBS at 4°C. The fixed cochlea was washed with PBS and then decalcified in 10% EDTA for two weeks with two weekly changes of solution. The cochlea was dehydrated, embedded in paraffin, and sectioned in a paramodiolar plane with a thickness of 6 μm, and every 10th section was mounted on a glass slide (a total of 10 slides per cochlea). Mounted sections were then processed for immunohistochemical analysis.

Brains and brain stems from each animal were cryoprotected in 30% sucrose in PBS at 4°C until the tissue settled to the bottom of the vessel, then they were embedded in Tissue-Tek (Sakura Finetek USA Inc. Torrance, CA) and serially sectioned at 18-20 μm in the coronal plane with a Thermo Cryotome (Thermo Fisher Scientific, Inc. Waltham, MA). One in ten slices from each brain and brainstem were mounted on gelatin precoated slides (10 total slides per brainstem and 20 slides per brain). The distance between two adjacent sections on each slide was approximately 200 μm. Sections were then processed for immunohistochemical analysis.

Quantification of spiral ganglion neurons and neurites in the cochlea

Two biomarkers, neurofilament (NF) light (68 kDa, NF-68) and heavy (200 kDa, NF-200) subunits, were used to examine cochlear nerve degeneration in blast-exposed rats. Cochlear sections were deparaffinized in xylene and rehydrated in successive concentrations of ethanol and distilled water. The sections were then washed with PBS, blocked in 1% bovine serum albumin (fraction V) and 1% normal horse serum or 1% normal goat serum in PBS, and blocked in 0.2% Triton X-100 (fraction V) in PBS. Permeabilized in PBS/T). Blocked and permeabilized sections were then mixed with mouse anti-neurofilament 68 (1:200, clone NR4, Sigma, St. Louis, MO, catalog #N5139) or chicken anti-neurofilament 200 (1:1000, EMD Millipore, Billerica, MA, catalog #AB5539) was incubated overnight at room temperature. After washing with PBS/T, biotinylated goat anti-chicken IgG or horse anti-mouse IgG (1:200, Vector Laboratories, Inc. Burlingame, CA) was applied to the slides for 1 hr at room temperature and treated with Vectastain ABC and DAB kit (Vector Laboratories, Inc. Burlingame, CA) for immunolabeling visualization. Immunopositive cells show brown reaction products at the site of the target epitope. Methyl green was used for nuclear counterstaining. Negative controls were performed by omitting the primary antibody. Toluidine blue was used to stain neurons in the SG of normal controls and blast-exposed rats to examine mean neuronal size and injury-induced depletion.

Tau immunohistochemical staining in brain and cochlea

The same immunohistochemical staining protocol as described above was used for Tau staining. Tissue sections were incubated with mouse anti-Tau-1 antibody (which recognizes all known electrophoretic species of Tau protein lacking phosphorylation at serine positions 195, 198, 199 and 202 (1:200, clone PC1C6, EMD Millipore, Billerica). , MA, catalog #MAB3420)), mouse anti-Tau 46 (which recognizes all six native isoforms of Tau (1:100, Sigma, St. Louis, MO, catalog #T9450)), mouse anti-phospholipid Tau antibody (1:250, clone AT8, Thermo Scientific, Waltham, MA, catalog #MN1020), or rabbit anti-oligo-Tau antibody (T22serum, 1:300, a kind gift from Dr. Rakez Kayed at the University of Texas Medical Branch, Galveston, TX, Hawkinset al. 2013).

Dual-labeling analysis of oligomeric Tau/NF-68 and Myosin VIIa/NF-200 in the cochlea after blast exposure

Cochlear sections were incubated with T22 antibody (1:200) and anti-Neurofilament 68 (1:200) or NF-200 (1:1000) and rabbit anti-Myosin VIIa (1:1000. Proteus Biosciences Inc., Ramona, CA, catalog #25-6790) was incubated at room temperature. After washing with PBS, sections were incubated with appropriate Alexa Fluor 488, 568, or 647 secondary antibodies (1:1000, LifeTechnologies, Co., Grand Island, NY) for 2 hours at room temperature, followed by DAPI labeling and mounting in antifade. in the culture medium. Images were acquired with a Zeiss LSM-710 confocal microscope.

Analysis of oxidative stress biomarkers in the cochlea after blast exposure

8-Hydroxy-2'-deoxyguanosine (8-OHdG) levels, a product of DNA oxidation, were measured as a biomarker to assess changes in oxidative stress-induced damage in cochlear neurons in blast-exposed animals . The same immunohistochemical staining protocol described above was used for 8-OHdG immunostaining. Tissue sections were incubated overnight with rabbit anti-8-OHdG antibody (1:100, Bioss antibodies, Woburn, MA, catalog #bs-1278R).

Quantification of biomarker immunostaining

Images were acquired with a BX51 Olympus microscope (SG and brain) or a Zeiss Axiovert 200m inverted fluorescence microscope (nerve fibers in helical lobes (SL)) and in the organ of Corti (OC). In the cochlea, images were acquired from the basal and middle gyri of all sections on each slide (nerve fibers in the SG, SL and the inner HC (IHC) area). The number of NF-68-, NF-200- or 8-OHdG-positive neurons was quantified using ImageJ software (National Institutes of Health). The percentages of NF-68 positive, NF-200 positive (weak or strong staining) and 8-OHdG positive neurons in SGs (positive staining/total number of neurons x 100%) were calculated and analyzed statistically. The density of NF-200-, Tau46-, AT8- or T22-positive nerve fibers in SL (number of nerve fibers/mm2) was also estimated using ImageJ software (Jensen et al., PLoS One 10.doi:10.1371/journal.pone. 0125160 (2015)). To detect SGN loss 21 days after blast exposure, the size of SGs at the middle and basal gyri of NC and blast-exposed rats was measured, and the relative density of SGNs (number of toluidine blue-stained neurons/mm2) was calculated and Analyzed statistically. To examine SGN size 21 days after blast exposure, the maximum diameter of toluidine blue-stained SGNs was measured in the cochlea of NC and blast-exposed rats (Bichler, 1984). Images were collected from the top, bottom, and middle gyri of cochlear slices, and the mean maximum diameter (μm) of SGNs was calculated and statistically analyzed. Only toluidine blue positive neurons were included in the analysis.

In the dorsal cochlear nucleus (DCN), images were acquired from the medial third (medial), medial third (medial) and lateral third (lateral) parts. In the inferior colliculus (IC), images were acquired from the central nucleus (CIC) of the IC. In AC, images were acquired from all layers (two images covering all layers on one section). Positive immunostained cells in these nuclei or regions were counted using a modified two-dimensional quantitative method (Du et al., PLoS One, e80138 (2013)). The total number of positive cells within each image was quantified using ImageJ software, and the density of Tau-positive cells in each group (number of positive cells/mm2) was calculated and statistically analyzed. Only dark brown stained cells were counted. Cell and neurite counts were performed by a technician who was unaware of the identity of the samples on each slide.

Statistical Analysis

One-way or two-way ANOVA (SPSS 14.0) and post hoc tests (Tukey HSD) were used to determine whether there were statistically significant differences between the three groups (NC, B and B/T) at each sampling interval. In these analyses, p-values less than 0.05 were considered statistically significant.

Neurodegeneration of the cochlea after blast exposure

Cochlear nerve degeneration typically begins within the fragile axonal neurites that innervate the HC, and typically progressively manifests as a loss of nerve fibers in the SL, ultimately leading to loss of the SGN (Jensen et al., PLoS One 10.doi:10.1371/journal.pone .0125160 (2015)). As a result, burst-induced neurogenesis in the OC was assessed by examining the relative density of ribbon synapses and NF-200-positive (type I) neurites in the IHC innervation area in both null and blast-exposed rats. damage. Dual immunolabeling analysis using antibodies against major ribbon markers (C-terminal aggregate protein 2, CtBP2) and markers for postsynaptic glutamate receptor plaques (GluR2/3) revealed that after injury At 7 and 21 days, synergistic blast-induced reductions in presynaptic and postsynaptic structures spanned the basal to middle gyrus of the OC (Figure 8, A-F). In contrast, low-frequency audio locations in the top gyrus do not appear to be affected by blast-induced synaptic loss. Independent quantification of these presynaptic and postsynaptic markers provided clear evidence that the severity of audio-specific synaptopathies was assessed from the basal to middle gyrus in blast-exposed rats (Fig. 9). In naïve rats, approximately 11-24 double-labeled synaptic foci per IHC were observed in the entire confocal section from the basal to middle gyrus of the OC. These numbers dropped sharply in untreated blast-exposed rats such that less than 50% of the normal number of postsynaptic elements was observed in IHC within the basal gyrus (ie, the 48 kHz audio region) 7 days after exposure. Ribbon synaptic density was also significantly reduced in the 8-32 kHz audio range in untreated rats at this assessment interval (38.1, 35.8 and 37.3% reduction in CtBP2puncta/IHC in the 8, 16 and 32 kHz regions, respectively, GluR2/3puncta/IHC decreased by 34.7, 28.8 and 34.7%, respectively). At 21 days post-exposure, in untreated animals, significantly reduced synaptic densities were still evident in the audio frequency range of 16-48 kHz (Figure 9). However, at this terminal time point, ribbon synapse density appeared to recover at a more apical 8 kHz audio position in these animals (Fig. 9), which may indicate the extent of localization of spontaneous reinnervation over time.

To track whether this blast-induced neuropathic response shifts over time to peripheral axonal retraction, longitudinal changes in relative neurite density were examined along IHC innervation areas at consecutive time points 24 hours, 7 days, and 21 days after blasting . In naive rats, approximately 8 distinct NF-200 positive neurites per IHC were observed in serial sections from the basal to middle gyrus of the OC (Figures 19A and 19D). Longitudinal analysis between blast-exposed rats showed a clear decrease in the number of NF-200-positive neurites along the IHC innervation zone at successive time points following destructive injury (Figures 19B and 19E). Formal quantification of these neurites confirmed a clear time-dependent loss of immunolabeling along this interface, with significant depletion first evident at 7 days post-blasting (Figure 19G). At the terminal sampling interval of 21 days, an average neurite loss of about 60% was observed near IHC in the basal gyrus of the OC of untreated blast-exposed animals. This degree of neurite loss is somewhat larger than would be expected from the average blast-induced synaptic loss at this time point measured along the same area, and may reflect a quantitative correlation with neurite density from tangential sections inherent reduction in resolution. Nonetheless, with only 1-2% of IHC losses recorded, these results indicate a marked decrease in neurotransmission in the peripheral auditory system in the mid- to high-frequency range in response to blast injury (Figures 9 and 19).

The relative density of NF-200-positive nerve fibers in bony SLs was investigated as a means to further evaluate progressive retrograde neurodegeneration in the cochlea of untreated blast-exposed rats. As shown in Figure 10, NF-200-positive nerve fiber bundles innervating the HC laterally from the SG were tightly packed in the bony SL of the control rats (Figure 10A). Although the apparent difference in the density of these nerve fibers was not evident at 24 hours and 7 days after blasting, a significant decrease in NF-200 staining intensity and nerve fiber density was first found in rats at 21 days after blast exposure (Figure 10B). Formal quantification of SL nerve fiber density over time supported these initial observations, revealing significant depletion that occurred between the 7- and 21-day intervals after blasting (Fig. 10D). These results indicate progressive retrograde neurodegeneration in addition to the initial loss of neurites along IHC synaptic connections, first observed at the 7-day sampling interval (FIG. 19G).

This temporal neurogenic trend was also shown in SG-containing neurons, such that the first apparent loss of NF-200 staining in the middle and basal gyrus of this cochlear nerve center was evident 21 days after blast exposure ( Figure 11B). In normal control animals, the majority of neuronal cell bodies were immunopositive for NF-200 labelling, with some cell bodies in each cross section showing strong immunohistochemical intensity (Figure 11A). These strongly labeled NF-200-positive neurons have previously been shown to represent type II neurons, therefore, neurons with light perinuclear body staining in the blank control were assumed to be type I neurons. To elucidate the type of degenerated neurons observed in the SG of blast-exposed animals, the percentages of weakly and strongly stained NF-200-positive somatic cells were independently calculated and statistically analyzed. At 21 days post blast, the percentage of weakly NF-200 immunopositive neurons was significantly reduced compared to normal control animals, whereas neurons with strong immunopositivity for NF-200 staining were significantly elevated as early as 7 days post blast exposure and It remained elevated for the remainder of the 21-day recovery period (Figures 11D and 11E). The increased perinuclear accumulation of NF-200 in the SGs of blast-exposed animals may represent cytoskeletal instability in type I neurons associated with trauma-induced demyelination and ongoing neurodegeneration.

Results from these spatial and temporal assessments together suggest that, following the initial blast-induced trauma, progressive cochlear neurodegeneration occurs in a retrograde fashion, culminating in a marked loss of neurofilament integrity in type I neurons in the SG. Without being bound by theory, the progressive destabilization of cochlear neurons could be complemented by a time-dependent increase in NF-68 immunostaining in SGs in response to blasting.

As shown in Figure 12, NF-68 immunolabeling of SGs from null rats resulted in a diffuse staining pattern with relatively few darkly stained neurons (ie, less than 0.5%, Figures 12A and 12D). However, in blast-exposed rats, an aberrant NF-68 immunoreactivity pattern was strongly observed 24 hours after blast and became progressively more pronounced over time (Figures 12B and 12D), such that 21 hours after bTBI day, the number of SGNs with strong NF-68 immunostaining surged to more than 7-fold that observed in the blank control. These results are consistent with the pattern of neurodegenerative responses shown by NF-200 immunolabeling (Figure 11). Despite its measurable somatic accumulation, no aberrant NF-68 immunoreactivity was detected in SG nerve fibers within SLs from either control or blast-exposed rats at any time point after blast exposure.

Supporting this apparent neurodegenerative response is the mean cell body of SG neurons in the cochlea of untreated, blast-exposed animals at day 21 post-traumatic in all three gyri compared with control Diameter was also significantly reduced (all p<0.001, Figure 13). However, SGN density analysis indicated that neuronal atrophy had not yet translated into a significant loss of neurons, as the total number of toluidine blue-positive SGNs was not significantly reduced at this terminal time point (all p>0.05).

Antioxidants reduce neurodegeneration in the cochlea after blast exposure

To specifically examine the temporal effect of this treatment regimen on the structural integrity of blast-exposed cochlear neurons, Applicants evaluated the above-mentioned ribbon synapses and nerves in a cohort of blast-exposed animals subsequently treated with HPN-07 and NAC Microfilament immunostaining pattern. Compared with untreated blast-exposed animals, antioxidant-treated rats exhibited no statistically significant loss of Ribbon synapses in the 8 and 16 kHz regions of the OC at 7 days post-traumatic (Figure 9, A and B). These treatment effects were temporarily extended to 21 days post-blast, as ribbon synapse density was indistinguishable from blank controls in the 16 kHz region in antioxidant-treated animals (Figure 8G-I and Figure 9C and D). A clear positive antioxidant treatment effect was also observed in the 32 kHz region at this terminal time point, although the magnitude of efficacy did not meet our criteria for statistical significance (ie p>0.05, Figure 9, C and D). HPN-07 and NAC treatment also reduced total neurite loss in IHC innervation areas in the middle and basal gyrus in response to neuropathic blast levels (Figures 19C and 19F). Furthermore, the progressive loss of synaptic neuritis processes observed in untreated rats was virtually absent in antioxidant-treated animals, such that at the terminal 21 day time point, treated relative to placebo There were no significant differences in the number of neurites per IHC in blast-exposed animals (Figure 19G).

This branch of therapeutic effect also manifested in a retrograde manner, as antioxidant intervention effectively counteracted the reproductive loss of nerve fibers in bony SLs caused by acute blast injury (Fig. 10C). In these treated animals, the density of SL nerve fibers was indistinguishable from the blank control at the terminal sampling interval of 21 days post blast (Fig. 10D). Therapeutic efficacy was also evident in the SG, where complete recruitment of weakly stained NF-200-positive type I neurons was detected throughout the experimental time course in blast-exposed rats administered HPN-07 and NAC (Figure 11). . This observation was complemented by a significant treatment-induced reduction in the number of NF-68 positive neurons in the SG at each sampling interval after blasting (Figures 12C and 12D). Furthermore, in contrast to the effect observed in untreated blast-exposed rats, the average size of SG neurons in all three gyri of animals treated with HPN-07 and NAC at a terminal sampling interval of 21 days post blast Indistinguishable from the blank control (all p>0.05, Figure 13). Thus, combined antioxidant intervention appears to effectively short-circuit blast-induced neurodegeneration in the peripheral auditory system.

Blast exposure induces hyperphosphorylation and oligomerization of Tau in spiral ganglion neurons

Generalized hyperphosphorylation and oligomerization of Tau in the hippocampus was induced in response to these blast conditions, both of which have been shown to trigger prion-like propagating waves of transcellular dysfunction independent of apparent persistent damage. Therefore, the accumulation of neurotoxic Tau in cochlear neurons that occurred was detected as a coincident sequelae of the explosion.

Cochlear assessment begins with the endogenous Tau antibody Tau-1, which recognizes the physiological isoform of Tau lacking phosphorylation at serine positions 195, 198, 199, and 202, and which can be used to differentiate physiology from pathology. However, the Tau-1 antibody did not show any detectable immunoreactivity with normal cochlear neurons or nerve fibers. Under physiological conditions, Tau can exist as one of six different isoforms produced by alternative splicing (Buée et al., Brain Res. Rev., 33, 95-130 (2000)). Furthermore, these isoforms undergo context-specific post-translational modifications (including differential phosphorylation) that modulate functional interactions with microtubules. Accordingly, Applicants used an alternative physiologically relevant antibody, Tau-46, which recognizes all six natural isoforms of Tau. Using this antibody, strong Tau-46 positive staining was observed in nerve fibers in SL and under HC in normal OC. Moderate Tau-46 immunolabeling was also observed in the cytoplasm of Pillar, Deiter and Hensen cells, and relatively weak staining was observed in IHC and outer HC (OHC). This cochlear distribution pattern is similar to that described in previous reports of physiological Tau and other microtubule-associated proteins such as α- and β-tubulin in this organ (Després et al. 1994; Oshima et al. 1992 ; Du et al., 2003; Slepecky and Ulfendahl 1992). In SGs, diffuse, positive Tau-46 staining was observed in the soma of neurons. However, approximately 10% of SGNs exhibited strong positive staining in null rats (Table 1). The spiral ligament and stria vascularis also exhibited diffuse Tau-46 staining (data not shown). These results indicate that, contrary to previous reports, the localization and distribution of Tau protein appears to be very widespread in the cochlea (Després et al. 1994; Oshima et al. 1992; Slepecky and Ulfendahl 1992), and that Tau is abundant in cochlear neurons and sensory epithelia The normal phosphorylation state of s may differ from that observed in hippocampal neurons (Du et al., 2016).

Table 1. Normal Tau staining in the auditory system after blast exposure and antioxidant treatment

Note: Numbers represent mean ± SEM; "NS" indicates no positive staining; "*, **, ***" indicate p<0.05, 0.01, 0.001 compared to NC, respectively; "treatment effect" refers to blast exposure Comparison between (B) and blast-exposed treated (B/T).

Somatic accumulation of normal tau protein is a hallmark of axonal microtubule instability in response to many acute and chronic neurodegenerative diseases (Kowall and Kosik, 1987; Wolfe, 2012). However, qualitative and quantitative assessment of the pattern of somatic Tau-46 immunoreactivity in the SGN of untreated blast-exposed rats showed that at any time point after blast exposure, compared with that observed in the blank control group, There were no detectable differences in total Tau levels (all p>0.05). Tau-46 immunoreactivity in SL was also measured at the terminal 21 day time point and statistically analyzed, the results of which showed a moderate increase in the number of Tau-46 strongly immunopositive nerve fibers (1846 ± 182.83/mm2, compared to 1462±158.66/mm2 in the blank control (p<0.05)). In blast-exposed rats treated with HPN-07/NAC, the density of Tau-46 positive nerve fibers (1313.98±128.23/mm2) was significantly reduced (p<0.01) compared to untreated blast-exposed rats And statistically indistinguishable from the blank control (p>0.05).

In pathological conditions, toxic insults, including oxidative stress, can lead to an imbalance in the activity of specific kinases and phosphatases, which leads to hyperphosphorylation of Tau at key microtubule regulatory sites, resulting in unbound neuronal soma increased levels of hyperphosphorylated Tau (Noble et al., 2013; Taniguchi et al., 2001). To discern whether SGNs are sensitive to this unstable stress response pattern in blast-exposed rats, Applicants immunolabeled cochleae from these animals and controls with the antibody AT8, which specifically recognizes hyperphosphorylated Tau organize. As shown in Figure 14, SGs of null rats were substantially unresponsive to immunolabeling with AT8 antibody (Figures 14A and 14D). However, blast exposure caused acute and chronic increases in somatic AT8 immunolabeling in SGNs, with peak immunoreactivity observed at day 7 of the post-blast sampling interval (Figures 14B and 14D, Table 2). However, levels of these neurotoxic variants appeared to decrease in the SGN on day 21 after blasting, suggesting that the detrimental effect of blasting on this microtubule-associated protein may be a transient phenomenon. The density of AT8-positive nerve fibers in the SL was also measured 7 days after blast exposure and statistically analyzed, at which point SGN exhibited statistically significant AT8 accumulation in untreated rats. However, the relative density of AT8-positive nerve fibers in the SL of blast-exposed rats was not significantly increased at this time point with or without treatment compared to the blank control group (169.17 ± 20.98) (untreated and treated 227.53±22.20 and 253.93±24.90/mm2 in rats, respectively) (all p>0.05).

Table 2. AT8 staining in auditory system after blast exposure and antioxidant treatment

Hyperphosphorylation of Tau is often a disease-causing precursor to Tau oligomerization, as the phosphorylation events that initially destabilize its microtubule-binding capacity lead to structural conformations that exhibit a propensity for self-association (Iqbal et al., 2013). This altered affinity pattern can lead to further Tau dysfunction as physiological isoforms of Tau are recruited to dead-end pathological complexes with hyperphosphorylated isoforms that enhance their neurodegenerative properties ( Takashima 2013). To investigate whether our blast exposure model also induces oligomerization of Tau in SGN, Applicants immunolabeled SG sections with antibody T22, which specifically recognizes pathological Tau oligomers, at longitudinal time points after blasting (Lasagna-Reeveset al., 2012). Similar to that observed with the AT8 antibody (Figure 14A), the vast majority of blank SGNs (>99%, Figure 15D) were not immunoreactive with the T22 antibody (Figure 15A). In contrast, novel and prevalent somatic T22 immunoreactivity was observed in the SGN of blast-exposed rats at all time points analyzed (Figures 15B and 15D, Table 3). The prevalence of this T22 immunolabeling pattern became more prevalent over time, in contrast to the trend observed with the AT8 antibody, which decreased after reaching peak levels 7 days post-blast (Figure 14D ). The density of T22-positive nerve fibers in the SL was also measured at the time point of maximal T22 accumulation in the SGN (ie, 21 days after blasting), and statistical analysis was performed. A statistically significant increase in T22-positive nerve fibers was also observed in the SL of untreated blast-exposed rats at this time point (516.99±15.38/mm2) compared to the blank control group (136.67±15.38/mm2). 58.17/mm2) (p<0.001). Persistence of this abnormal T22 SGN immunolabeling pattern in blast-exposed rats indicates a persistent or progressive pathology of this auditory nerve center.

Table 3. T22 staining in auditory system after blast exposure and antioxidant treatment

The T22 profile observed in blast-exposed rats is reminiscent of the NF-68 longitudinal immunolabeling pattern described above for this cohort. This prompted us to determine whether these complementary trends manifest as simultaneous pathophysiological responses in the same degenerating SGN. To this end, SG tissue sections from blast-exposed rats were co-incubated with antibodies against NF-68 and oligomeric Tau, and then the potential co-localization of these two pathological epitopes was assessed. An example of this immunofluorescence analysis is shown in FIG. 16 . Based on these analyses, it was evident that, although T22 immunolabeling was generally more prevalent than NF-68 at the acute and chronic sampling intervals, multiple T22-positive neurons were co-labeled with NF-68 antibodies at each time point (Fig. 16). The size and shape of these double-labeled neurons were consistent with type I SGNs. These results suggest that destabilization of progressive neurotoxicity of tau function and destabilization of neurofilaments are relevant events in degenerated SGN in blast-exposed rats.

Antioxidant treatment reduces blast-induced accumulation of neurotoxic Tau variants in spiral ganglion neurons

To determine whether the apparent therapeutic effect of HPN-07 and NAC intervention on cochlear neurodegeneration revealed by differential neurofilament staining could extend to Tau dysfunction in the SG, Applicants immunolabeled with AT8 and T22 antibodies at each sampling interval Relevant tissue sections from antioxidant-treated blast-exposed rats for comparison with control and untreated blast-exposed cohorts. As shown in Figure 14 (Panels C and D), post-acute injury intervention using the combined regimen of HPN-07 and NAC reduced the expression of hyperphosphorylated Tau at all time points examined. Of particular note, when AT8 levels peaked in the untreated blast cohort, a significant reduction in the number of AT8-immunopositive neurons was observed in treated rats with a 7-day sampling interval (Figure 14D and Table 2). However, at this time point, antioxidant treatment did not reduce the number of AT8-positive neurons to the levels observed in the blank controls, which may underline the saturable or oxidative stress of blasting aberrant Tau phosphorylation in SGs independent effect.

Complementary analysis with oligomeric Tau antibody revealed a more pronounced effect of HPN-07/NAC intervention in neurons in the SG of blast-exposed animals. Under these conditions, antioxidant intervention significantly and effectively blocked the pathological increase in T22 immunolabeling observed in the cell bodies of untreated blast-exposed rats over the entire time course of the study ( Figure 15, Panels C and D, Table 3). This positive treatment effect was also observed in SL, where T22-positive nerve fiber density (193.53 ± 21.37/mm2) was significantly less than in untreated blast-exposed rats (516.99 ± 58.17/mm2, p<0.001), and Statistically indistinguishable from the blank control (p>0.05) at the terminal sampling interval. Combined with the positive therapeutic effect of preservation of NF-200 staining in SGNs (Figure 11) and the attenuation of pathological NF-68 accumulation (Figure 12), these results suggest that this combined antioxidant regimen in blast-exposed animals has previously implicated Therapeutic effects can be extended to include inhibition of neurodegeneration and progressive Tau dysregulation in the peripheral auditory system.

Antioxidants reduce somatic Tau accumulation between neurons in the auditory cortex of blast-exposed rats

While combined HPN-07/NAC treatment significantly improved persistent signs of bTBI-related tau dysfunction in the peripheral auditory system, this experiment sought to determine whether repeated blast exposure also elicited the effects of therapeutic antioxidant intervention in the central auditory system. Offset pathological changes in Tau. To this end, tissue sections from anterior ventral cochlear nucleus (AVCN), posterior ventral cochlear nucleus (PVCN), DCN, IC and AC were evaluated for immunocytological evidence of Tau dysregulation in response to blasting. In contrast to cochlear tissue, neurons within the central auditory pathway were diffusely immunoreactive with conventional physiological Tau-1 antibodies in naïve rats (FIG. 17A). This observation is consistent with Applicants' previous studies on Tau immunoprofiling in neurons of the hippocampus (Du et al., 2016). In the blank control, Tau-1 positive neurons were distributed in all layers of the DCN and in all regions of the VCN and IC (Table 1), with a very low frequency of strong somatic immunoreactivity. Contrary to our previous assessment of hippocampal neurons, blast exposure did not cause significant changes in somatic Tau-1 staining in the AVCN or DCN, and only in the PVCN of untreated blast-exposed animals, 7 days after blasting A transient increase in somatic Tau-1 immunoreactivity was detected at the sampling interval of 100% (p<0.05, Table 1).

In blank control ACs, Tau-1 positive neurons were mainly located in the deep neuronal layer. However, after blast exposure, Tau-1 positive neurons were also observed in the middle layer. As graphically summarized in Figure 17, significantly more Tau-1 positive somatic cells were observed in neurons in the AC at all time points after blast exposure. This blast-induced effect appears to be biphasic, as the number of dark somatic Tau-1-stained neurons in the AC further increased 21 days after blast following an initial plateau between 24 hours and 7 days after blast Elevated, perhaps reflecting acute and progressive dysregulation of normal tau function.

Examination of central auditory tissue sections with AT8 antibody showed no significant induction of blast-induced hyperphosphorylation in AVCN, PVCN or IC (Table 2). A slight transient increase in somatic AT8 immunolabeling was observed in neurons of the DCN on day 7 post blast (p<0.05, 2.8-fold increase), with no apparent detectable change at the 24-hour or 21-day time points (Table 1). 2). In primary AC, a more significant (p<0.01, 4.76-fold increase) but still transient increase in AT8 immunoreactivity was observed 7 days post blast relative to that detected in DCN. However, this pathological staining pattern appeared to resolve at the 21-day sampling interval.

T22 immunolabeling exhibited a similar immunolabeling pattern to that observed with the AT8 antibody in the central auditory pathway of blast-exposed animals. No T22-responsive neurons were detected in the AVCN, PVCN, DCN, or IC of null animals (Table 3). Furthermore, our bTBI model failed to induce a significant increase in somatic T22 immunoreactivity in these central auditory regions of the brain over the experimental time course studied (Table 3). In contrast, the prevalence of T22-positive neurons in the AC was significantly elevated 7 days after blasting (p<0.001, a 100-fold increase). However, this abnormal immune labeling pattern was significantly reduced at the terminal (day 21) time point of the study (Table 3). Taken together, these results reveal that bTBI induces persistent, if not progressive, somatic accumulation of physiological Tau in neurons of AC, which is incompatible with transient but transient but not progressive accumulation of hyperphosphorylated and oligomeric Tau in this central auditory center. Delayed (7 days after detonation) accumulation combined.

When rats were administered a combination antioxidant (HPN-07/NAC) treatment regimen after blasting, somatic physiological Tau accumulation (i.e., somatic Tau) in neurons of primary AC was observed at the 7 and 21 days post blasting time points. -1 reactivity) was significantly reduced (Figure 17, Table 1). Positive treatment effects were most pronounced at the terminal 21-day time point, where the relative prevalence of somatic Tau-1-positive neurons was approximately two-fold lower than that observed in untreated blast-exposed rats, indicating definitive treatment Specific effect (p<0.001). However, in contrast to the observed effects on somatic Tau-1 accumulation, this therapeutic effect did not extend to the abnormal AT8 or T22 immunoreactivity patterns observed in DCNs and/or ACs, as antioxidant treatment appeared to have a significant effect on blasting The delayed but transient increases in Tau hyperphosphorylation and oligomerization observed in these central auditory nuclei of exposed animals had no apparent effect (Tables 2 and 3). Thus, the pathological and therapeutic response patterns of neurotoxic Tau accumulation in peripheral and central auditory pathway neurons of blast-exposed rats are markedly different, possibly reflecting their relative anatomical location and response to blast-induced reproductive oxidative stress and neurodegeneration background susceptibility.

Antioxidants reduce oxidative stress between neurons in the spiral ganglia of blast-exposed rats

To confirm that blast-induced oxidative stress does, in fact, contribute to the pathophysiological responses associated with cochlear neurodegeneration, Applicants immunolabeled SGN tissue 24 hours after blasting with 8-OHdG, an organism responsible for oxidative DNA damage marker organisms (Valavanidis et al., 2009). Intense 8-OHdG positive staining was observed in the nuclei of SGNs of untreated blast-exposed rats, indicating a damaging level of oxidative stress in these neurons (Figure 18B). As graphically summarized in Figure 18D, significantly higher numbers of 8-OHdG positive neurons were observed in the SGs of these animals compared to the control (p<0.001). The prevalence of SGN with strong 8-OHdG immunoreactivity was significantly reduced in animals treated with the combined antioxidant regimen (p<0.001). These results provide direct evidence for oxidative stress as a contributing, if not a major, factor in blast-induced neurodegeneration in SG, and the positive therapeutic effect of antioxidant treatment on reducing stress responses.

Antioxidants reduce loss of auditory function in blast-exposed rats

The ABR results of this study have been described in detail in our previous report (Ewert et al., 2012) and are summarized in Figure 20. Overall, significant changes in ABR thresholds were observed in untreated blast-exposed animals at all time points following blast exposure. Compared to the untreated blast-exposed group, the ABR threshold shift in the HPN-07/NAC-treated group was reduced by approximately 10 dB at 24 hours post blast and 21 dB at 7 and 21 days post blast (all p<0.001). Significant recovery (p<0.01 or 0.001) of changes in ABR thresholds in the antioxidant-treated group was measured at all frequencies tested (2-16KHz) at 7 and 21 days after blast exposure. Consistent with the observed therapeutic efficacy against progressive SG neurodegeneration, these ABR results reflect the well-defined positive properties of HPN-07 and NAC for interrupting the ongoing pathophysiology that results in a progressive loss of auditory function in blast-exposed rats reaction.

further analysis

While our blast model has been shown to induce significant and permanent changes in ABR thresholds, indicating diminished sensory nerve function, only 1% was observed in the middle and basal gyri of untreated blast-exposed rats 21 days post-injury to 2% IHC loss, suggesting that the degree of neurodegeneration was not apparent in these animals (Ewert et al. 2012; Du et al., 2013 and data not shown).

Indeed, starting 7 days after injury, we observed a significant decrease in the number of neurites in the IHC innervation areas along the middle and basal gyrus of the OC in untreated blast-exposed rats, and at the end of the study Significant loss of more than half of the original neurite population was measured in these regions at the last 21 day time point (Figure 19). These results correlate with significant blast-induced loss of ribbon synapses between IHCs along the width of the region at the terminal 21 day posttraumatic time point (Figures 8 and 9), indicating a marked reduction in peripheral auditory signaling to the brain . In these animals, the number of peripheral axons in the bony SL was not significantly reduced until 21 post-blast (Figure 10). These results are consistent with a gradual but progressive axonal retraction of IHC synapses in response to loss of our blast exposure paradigm, similar to that recorded in mice exposed to acute auditory trauma (Jensen et al. 2015) . During this period, pathological NF-68 staining remained significantly elevated in SGN somatic cells (Figure 12), indicating persistent dysfunction. On the other hand, a marked imbalance in the normal NF-200 immunolabeling pattern of SGN somatic cells was not observed until 7 days after blasting, with a marked decrease in the number of neurons with a type I immunoreactivity pattern first evident on day 21 post-injury (Figure 11). These results appear to suggest that pathological NF-68 accumulation is a more sensitive or epistatic marker for the pathology of ongoing blast-induced neuropathy than loss of NF-200 immunoreactivity in SGN.

The pattern of progressive pathophysiological responses observed in the SGN in response to our blast injury model is characteristic of neurodegeneration associated with mild blast-induced TBI (mTBI) and other clinically relevant neuropathies (Goldstein et al., 2012; Sajja et al., 2015; Walker and Tesco, 2013). The strength of our blast overpressure (14 psi) model may play a key role in the timing and extent of blast-induced neurodegeneration within the SG. In a related study in mice, Cho and colleagues demonstrated that while no loss of SG neurons was observed in animals exposed to 94 (13.63 psi) or 123 (17.84 psi) kPa blasts, 181 kPa (26.25 psi) The detonation of sigmoid induced significant SGN loss as early as 7 days after trauma (Cho et al., 2013). In the present study, the results of toluidine blue and NF-200 staining indicated that there was no significant neuronal loss in the SG at the terminal experimental time point (21 days). Nonetheless, the persistent pathological accumulation of NF-68 and neurotoxic Tau variants in SGNs that we observed throughout our study is consistent with a degree of blast-induced neuropathy that may ultimately contribute to this peripheral Significant decline in neuronal population. Consistent with this rationale, we observed a statistically significant reduction in the mean somatic diameter of SGNs in the cochlea of untreated blast-exposed rats 21 days after blast, indicating ongoing neuronal atrophy in these animals (Fig. 13) (Raff et al., 2002). In addition, the mTBI model employed in this paper is more likely to mimic those encountered by military personnel, thereby providing potential insights into the progressive spatiotemporal neurodegeneration commonly seen in veterans (McKee and Robinson, 2014; Yankaskas, 2013).

mTBI induced by explosive overpressure exposure is known to induce prolonged oxidative stress (Abdul-Muneer et al., 2013; Kochanek et al., 2013; Readnower et al., 2010). We therefore examined the therapeutic effect of a post-traumatic intervention on the pathophysiological response in blast-induced OC with an antioxidant formulation consisting of the classical antioxidant N-acetylcysteine and a free radical spin trap (HPN-07) . We found that this therapeutic strategy was effective in preventing direct manifestations of oxidative stress arising from the pathophysiological response of our blast injury model (Figure 18), and in preventing acute and chronic loss of NF-200-positive neurites in IHC innervation areas and nerve fiber loss in the SL in blast-exposed rats (Figure 19 and Figure 10). This therapeutic efficacy also translated into efficient ribbon synaptic preservation in the 16 kHz region of the OC, and an indication of a positive treatment effect in the 32 kHz region (Figures 8 and 9). In addition, HPN-07/NAC intervention also significantly reduced pathological NF-68 accumulation in somatic cells of SGN and neuronal imbalance of NF-200 immunostaining, and attenuated blast-induced average somatic cells in these neuronal populations A reduction in diameter (Figures 11-13). It is unclear whether these positive therapeutic effects are a direct effect of HPN-07 and NAC on reducing oxidative stress within neurons and neurites, an indirect protective effect through sustained HC viability, or a combination of both. However, as noted above, only 1-2% IHC loss was observed over the entire time course of our study (Ewert et al. 2012; Du et al., 2013). Given the relatively early loss of IHC neurites in untreated blast-exposed rats, the therapeutic effect observed in antioxidant-treated animals advocates direct protection of SGN neurites. Indeed, the more robust treatment effect observed along the width of the OC to maintain average peripheral axonal density relative to preservation of ribbon synaptic integrity may suggest that antioxidant intervention slows or prevents further axonal retraction, a treatment outcome that Intrinsic or therapeutic reinnervation strategies can be enhanced (Tong et al., 2013; Wan et al., 2014).

Aberrant phosphorylation and aggregation of the microtubule-associated protein Tau are both induced and enhanced by oxidative stress (Melov et al., 2007; Mondragón-Rodríguez et al., 2013). Furthermore, in many cases acute restraint of Tau function can lead to chronic cytoskeletal destabilization that propagates in a transcellular manner, as pathological Tau oligomers from degenerating neurons recruit functional Tau as neurotoxic oligomers (Clavagura et al., 2013; Guo et al., 2011). Based on these observations and our previous study of tau in the CNS of blast-exposed rats (Du et al., 2016), we examined whether peripheral and central auditory pathways show tau potentially contributing to sensorineural hearing loss Evidence of a tauopathic response.

Using our bTBI model, we found that blast exposure caused acute Tau hyperphosphorylation in the soma of SG neurons of untreated rats, which peaked at 7 days post trauma (Table 2 and Figure 14). This pathological response was reflected by the accumulation of oligomeric Tau inclusion bodies that remained elevated throughout the experimental time course of our study (Table 3 and Figure 15). Taken together, these results suggest that blast-induced Tau dysfunction is a coincident molecular sequelae of neurofilament instability and neurodegeneration in OC, suggesting a widespread and persistent negative impact on cytoskeletal integrity among SG neurons.

Based on the fact that the pathological response patterns of NF-68 and Tau staining in our blast model are closely related to each other and that neurofilament and Tau dysfunction are important in neurodegenerative diseases such as chronic traumatic encephalopathy, amyotrophic lateral sclerosis, and Alzheimer's disease), we examined whether these two pathological markers co-exist in the somatic cells of degenerated SGNs after blasting (Dekosky et a., 2013; Lin and Schlaepfer, 2006; Nguyenet al., 2001; Schmidt et al., 1990; Vickers et al., 1994). We found that NF-68 accumulation and Tau oligomerization are in fact colocalizable sequelae in SGNs, but based on their relative prevalence, Tau oligomerization may be the destabilizing factor of neurofilaments in this pathological setting Epistatic precursor (Figure 16).

Although oxidative stress is a controlling factor in the induction and persistence of Tau hyperphosphorylation and oligomerization in the CNS, little is known about the clear physiological mechanisms linking these pathological responses (Alavi Naini and Soussi-Yanicostas, 2015) . Nonetheless, our findings suggest that early intervention with HPN-07 and NAC has a clear therapeutic effect on these neurotoxic manifestations of Tau in SG neurons and their peripheral nerve fibers (Figures 14 and 15), which is consistent with blast-induced oxidation The theory that stress may also drive this tau response in the peripheral auditory system is largely consistent. Of particular note, our combined antioxidant formulation significantly reduced both acute and chronic oligomeric Tau accumulation in SGs following acute blast exposure (Figure 15). Since Tau oligomers are widely considered to be the major infectious neurotoxic agents in many tau diseases, including Alzheimer's disease (Lasagna-Reeves et al., 2010 and 2012; Usenovic et al., 2015; Violet et al. , 2015), thus the ability of early HPN-07 and NAC intervention to inhibit its formation in the SGN may confer significant long-term protection against progressive neurodegeneration in the cochlea.

Like Tau, NF-68 is susceptible to oxidative stress-induced hyperphosphorylation and oligomerization, and there is evidence that aggregates of dysfunctional NF-68 can act as non-physiological chaperones that promote Tau oligomerization (Ishihara et al., 2001). Previous in vitro studies have shown that classical antioxidants such as NAC and HPN-07-related radical spin traps such as α-phenyl N-tert-butylnitrone (PBN) are able to protect native Tau and NF -68 is protected from oxidative stress-induced aggregation (Kimet al., 2003 and 2004; Olivieri et al., 2001). In our neuropathic blast study, the consistent ameliorating effects of HPN-07 and NAC on somatic accumulation of NF-68 and neurotoxic Tau variants in SGN suggest that post-traumatic intervention using this therapeutic agent is also possible in vivo Short-circuit these interconnected patterns of molecular stress responses.

Although blast exposure resulted in pathological Tau immunostaining in the peripheral and central auditory systems, we found that neurons in the SG and AC were more susceptible to this maladaptive response than those in the CN and IC (Valiyaveettil et al. , 2012). However, antioxidant intervention was more effective in attenuating abnormal Tau modifications in the peripheral auditory organs than in the central auditory system, where the therapeutic effect was limited to the somatic accumulation of physiological Tau rather than hyperphosphorylated or oligomeric Tau accumulation (Tables 1-3 and Figure 17). This difference may reflect differences in the relative penetrance of HPN-07 and NAC across the blood-brain barrier versus the blood-cochlear barrier or differences in the way pathophysiological responses originate in these tissues. The results showed that the same combination antioxidant regimen was sufficient to interrupt these tauopathy responses in hippocampal neurons, suggesting that, at least in this subcortical region of the brain, the drug reaches sufficient concentrations to mitigate blast-induced tau dysfunction (Du et al., 2016). Therefore, prolonged treatment or higher doses of these antioxidants may be required to more effectively combat pathological Tau accumulation in the central auditory system.

Taken together, our results demonstrate that blast exposure-induced mTBI induces progressive retrograde neurodegeneration in the peripheral auditory system, and that early intervention with HPN-07 and NAC provides important protection against this outcome. Furthermore, the ability of these antioxidants to prevent widespread Tau dysfunction and pathological aggregation in the SGN further underlines their long-term ameliorating benefits for limiting reproductive neurotoxicity in the inner ear.

Example 3

found that noise-impaired animals treated with 2,4-disulfonyl α-phenyl-tert-butylnitrone (HPN-07) exhibited significantly higher IHC neurite populations relative to untreated controls, and that HPN -07 treatment uniquely caused progressive functional recovery in these animals, suggesting ongoing reinnervation. Therefore, we investigated whether HPN-07 efficiently induced neurite outgrowth and synaptogenesis.

HPN-07 was tested in three in vitro neurogenic models (PC12 cells, and co-cultures of cochlear spiral ganglion explants, and SGN explants with attached hair cells (HC). Experimental data from these analyses demonstrate that: (1) HPN-07 enhances NGF-induced neurite outgrowth in PC12 cell line; (2) HPN-07 promotes neurite outgrowth in hairless spiral ganglion explants (3) HPN-07 reversed excitotoxic ribbon synaptic loss and increased neurite density along the base of IHC in SGN-HC co-cultures following kainic acid (KA)-induced agonistic trauma.

The expansion of HPN-07 to further characterize and optimize the pro-neurogenic properties of HPN-07 in vitro, and to translate these findings into therapeutic results from in vivo animal experiments, aims to test HPN-07 treatment for regenerating IHC-SGN synapses in vivo efficacy, using an acoustic overexposure paradigm that has been shown to cause a minimal threshold shift in the IHC of the cochlea but cause permanent afferent blockade. The experimental results showed that: (1) HPN-07 enhanced BDNF-induced and NT-3-induced neurite outgrowth in SGN explants; (2) HPN-07 (formulated with NAC, HPN-07/NAC) prevented Noise-induced hearing loss in rats; (3) HPN-07/NAC reverses noise-induced excitotoxic loss of IHC ribbon synapses in rats; and (4) HPN-07/NAC restores noise-induced hearing loss in rats ABR amplitude due to noise.

We have demonstrated that HPN-07 enhances BDNF-induced and NT-3-induced neurite outgrowth of type I axons in spiral ganglion tissue explants in a dose-dependent manner in vitro. BDNF and NT-3 are intrinsic neurotrophic factors in the inner ear and are essential for the development and survival of the cochlear spiral ganglion (SGN). As shown in Figures 24-27, exogenous BDNF and NT-3 at 10 [mu]M elicited significantly of neurite outgrowth (Figure 24B and Figure 26B). In addition to BDNF or NT-3, HPN-07 further increased the number and extent of nerve fibers (Figures 24C-D and 26C-D). Compared to NC, neurite length was not altered by any of the treatments (Figures 25 and 27).

In vivo synaptogenesis experiments were designed as follows. SpragueDawley rats weighing 250-300 g (Charles River) were divided into three groups: NC (no noise), noise alone (treatment with saline), and noise + HPN-07/NAC (with 300 mg/kg of HPN-07/ NAC treatment). Rats were exposed to octave band noise (8-16 kHz) at 110 dB for 2 hours. Treatment was initiated 24 hours after noise exposure, with 5 doses of treatment administered over 3 days. Two weeks after treatment, cochlear samples (hair cell and synapse counts) were collected and analyzed. Hearing was tested by ABR before the noise and retested 1 and 15 days after the noise.

Short-term hearing loss detected by temporal threshold shift (TTS) measured 1 day after noise was similar between untreated and treated (Fig. 21A), implying that noise caused noise in both groups similar damage. Continued HPN-07/NAC treatment prevented permanent noise-induced hearing loss measured after 15 days of noise exposure (Figure 21B). TTS is immediate hearing loss after exposure to noise that recovers over time. Not all TTS goes away, and the final hearing loss that remains is called permanent hearing loss. Hair cell loss was minimal after noise exposure, occurring only in the basal region of the cochlea (Figure 22). There were no significant differences between the three groups studied.

In Sprague Dawley rats, one-shot noise (8-16 kHz, 2 hours, 110 dB) resulted in 30-40% loss of synapses at high frequencies. HPN-07/NAC treatment started 24 hours after injury reversed the injury. Synapses are "connectors" that transmit sound information decoded by sensory hair cells to the brain (see red and green dots in Figure 23). Loss of synapses has been implicated in difficulty understanding speech in noisy environments, age-related hearing loss, tinnitus, and the like.

As shown in Figure 34, the auditory brainstem response (ABR) wave I amplitude and V/I amplitude ratio were significantly reduced at 2 weeks after 80 dB SPL exposure compared to the pre-noise baseline in the non-treatment group. In contrast, ABR wave I amplitude and V/I amplitude ratio did not change in the HPN-07/NAC-treated group. ABR wave V amplitudes did not change in either the treated or untreated groups. The ABR test objectively measures auditory neural responses in response to sound, which provides information about the inner ear (cochlea) and the brain pathways of hearing. It is often used in clinics and research. ABR wave I is thought to arise in the peripheral or distal portion of the auditory nerve. ABR wave V is thought to be generated in the lateral lemniscus and hypothalamus of the brainstem. Wave I amplitude was reduced in the untreated group, indicating that auditory input to the inner ear was reduced due to loss of synapses, so wave V amplitude and V/I amplitude ratio increased to compensate for the brainstem, indicating increased neural activity in the central nervous system, it was It is suggested that it can cause tinnitus. With HPN-07/NAC treatment, the recovery of wave I amplitude was consistent with the recovery of synaptic loss (see Figure 34), so wave V was unchanged. Our results demonstrate that HPN-7/NAC can restore noise-induced hearing impairment and that ABR wave amplitudes can be used to monitor synaptic loss and drug treatment effects.

Taken together, our results demonstrate that HPN-07 represents a safe pharmacological means to regenerate lost ribbon synapses in the inner ear, a promising candidate for the treatment of cochlear synaptic disease and its associated prevalent clinical manifestations such as difficulty understanding noisy environments speech, presbycusis, hyperacusis, and tinnitus) offer a promising non-invasive alternative.

Example 4 - Established/chronic hearing loss

As shown in Figure 28, a pilot study of established hearing loss was designed. A permanent threshold shift is established through wild explosion damage. Treatment started 4 weeks post-injury with HPN-07 plus NAC administered at 300 mg/kg twice daily for 14 days. Figure 28 further shows ABR threshold improvement at 14 weeks post injury (8 weeks post treatment) compared to pre-treatment.

As shown in Figure 29, NHPN-1010 treatment (HPN-07 plus NAC) restored IHC ribbon synapse number in an established chronic hearing loss model. NHPN-1010 treatment was initiated 4 weeks after injury and administered daily for 14 days. Ribbon synapse counts recovered significantly 8 weeks after injury.

As shown in Figure 30, NHPN-1010 treatment resulted in restoration of ABR wave I amplitude in an established model of chronic hearing loss. The magnitude of responses to 4 kHz and 8 kHz stimuli at 80 and 70 dB SPL in the placebo group decreased with blast exposure and continued to decrease over time. The reduction in amplitude in response to 8 kHz stimulation at 80 kHz SPL fully recovered to pre-treatment levels 8 weeks after NHPN-1010 treatment.

Furthermore, as shown in Figure 31, Ribbon synapses were reduced after blasts at 16, 32, 48 kHz and were maintained/recovered with NHPN-1010 treatment (HPN-07 plus NAC). Furthermore, as summarized in Figure 32, NHPN-1010 treatment (HPN-07 plus NAC) was able to improve blast effects on primary auditory afferent neurons - reducing retrograde neurodegeneration.

Example 5 - Tinnitus Biomarkers

The blast (B) group received one shock tube blast (10 psi). The blast/therapy (B/T) group further received a total of 5 doses of HPN-07/NAC. Tissues were harvested 8-9 weeks after blast exposure. Animals in each experimental group (6-8 rats/time point) were euthanized and perfused intracardially first with saline and then with 0.1 M phosphate buffered saline (PBS, pH 7.2) containing 4% paraformaldehyde . After removal of the cochlea, brain and brain stem, they were fixed in the same fixative (overnight for cochlea, one week for brain). The fixed cochlea was washed with PBS and then decalcified in 10% EDTA for two weeks with two weekly changes of solution. The cochlea was dehydrated, embedded in paraffin, and sectioned in a paramodiolar plane with a thickness of 6 μm, and every 10th section was mounted on a glass slide (a total of 10 slides per cochlea). Mounted sections were then processed for immunohistochemical analysis. Brains and brain stems from each animal were cryoprotected in 30% sucrose in PBS at 4°C until the tissues settled to the bottom of the vessel, at which point they were embedded in Tissue-Tek (Sakura Finetek USA Inc. Torrance). , CA) and serially sectioned at 18-20 μm in the coronal plane with a Thermo Cryotome (Thermo Fisher Scientific, Inc. Waltham, MA). One in ten slices from each brain and brainstem were mounted on gelatin precoated slides (10 total slides per brainstem and 20 slides per brain). The sections were then washed with PBS, blocked in 1% bovine serum albumin (fraction V) and 1% normal horse serum or 1% normal goat serum in PBS, and blocked in 0.2% Triton X-100 (PBS) in PBS /T) medium permeabilization. Blocked and permeabilized sections were then incubated with primary antibody overnight at room temperature. After washing with PBS/T, biotinylated secondary antibodies (1:200, Vector Laboratories, Inc. Burlingame, CA) were applied to the slides for 1 hour at room temperature, and the slides were washed with Vectastain ABC and DAB kits (Vector Laboratories, CA). Inc. Burlingame, CA) for immunolabeling visualization. Images were acquired with a BX51 Olympus microscope for biomarker quantification. For fluorescent immunolabeling, slice the sections with the appropriate Alexa Secondary antibodies (1:1000, Life Technologies, Co., Grand Island, NY) were incubated at room temperature for 2 hours before DAPI labeling and mounting in anti-fade medium. Images were acquired with a Zeiss LSM-710 confocal microscope.

Activity-regulated cytoskeleton-associated protein (ARC), also known as Arg3.1, is a plasticity protein. Decreased ARC in the central auditory system is associated with tinnitus. Figure 35 shows ARC immunostaining in the central auditory system. Blast exposure decreased ARC in AC, IC and DCN. HPN-07/NAC treatment normalized ARC expression in AC, IC, and DCN compared to the blast group without treatment.

Growth-associated protein 43 (GAP-43) is a membrane-associated phosphoprotein located in axonal growth cones. It is a marker of axonal growth, synaptogenesis and synaptic remodeling. Figure 36 shows GAP-43 immunostaining and western blotting in the central auditory system. Blast exposure upregulated GAP-43 in AC, IC and DCN. HPN-07/NAC treatment normalized GAP-43 expression in AC, IC, and DCN compared to the blast group without treatment.

GABAA receptors are ionotropic receptors and ligand-gated ion channels. Its endogenous ligand is gamma-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system. Figure 37 shows GABAA receptor alpha 1 (GABAAR alpha 1 ) immunostaining in DCN. Figure 38 shows GABAARα1 (red) and GAD67 (green) co-labeling in DCN. GAD67 is a biomarker of inhibitory neurons, indicating that GABAARα1-positive cells are inhibitory neurons.

Glutamate receptor 2 (GluR2) is an ionotropic receptor for AMPA, which is an excitatory neurotransmitter in the central nervous system. Overstimulation of glutamate receptors causes neurodegeneration and neuronal damage through excitotoxicity. Figure 39 shows GluR2 immunostaining in DCN.

Blast exposure upregulates the expression of GABAA and glutamate receptors in DCN, suggesting reorganization of inhibition and excitation in DCN. Compared with the blast group without treatment, HPN-07/NAC treatment normalized the expression of GABAA and glutamate receptors in DCN, restoring the balance between inhibition and excitation in DCN.

Transient receptor potential cation channel subfamily V member 1 (TRPV1), also known as capsaicin receptor and vanilloid receptor 1, is activated by high temperature, acidic conditions, capsaicin, and stimulatory compounds. Upregulation of TRPV1 in SG is associated with tinnitus. Figure 40 shows TRPV1 immunostaining in SGs. Blast exposure upregulated TRPV1 in the SG. HPN-07/NAC treatment normalized TRPV1 in SGs compared to the blast group without treatment.

As used herein, the singular terms "a," "an," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, unless the context clearly dictates otherwise, reference to a compound may include multiple compounds.

As used herein, the terms "substantially," "substantially," and "about" are used to describe and explain small variations. When used in conjunction with an event or circumstance, these terms can refer to both instances in which the event or circumstance occurs exactly as well as instances in which the event or circumstance is close to occurring. For example, these terms may refer to less than or equal to ±10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

Additionally, amounts, ratios, and other numerical values are sometimes presented in a range format. It is to be understood that this range format is used for convenience and brevity, and should be flexibly construed to include the numerical values expressly designated as the upper and lower limits of the range, but also to include all individual values or subranges subsumed within that range, As if each value and subrange were specified explicitly. For example, ratios in the range of about 1 to about 200 should be understood to include the expressly recited upper and lower limits of about 1 and about 200, but also include individual ratios, such as about 2, about 3, and about 4, and subranges, such as about 10 to about 50, about 20 to about 100, and so on.

From the foregoing description, it will be apparent to those skilled in the art that various substitutions and modifications of the invention disclosed herein can be made without departing from the scope and spirit of the invention. The invention illustratively described herein may be practiced without any element, limitation, not specifically disclosed herein. The terms and expressions that have been used are to be used as terms of description rather than limitation, and their use is not intended to exclude any equivalents or parts of the features shown and described, but it is recognized that in the present invention Various modifications can be made within the range. Therefore, it should be understood that while the present invention has been described in terms of specific embodiments and optional features, modifications and/or variations of the concepts disclosed herein may be employed by those skilled in the art, which modifications and variations are considered to be within the scope of the present invention Inside.

Equivalent

It should be understood that while the present disclosure has been described in conjunction with the above-described embodiments, the foregoing description and examples are intended to illustrate, and not to limit, the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The embodiments illustratively described herein may suitably be practiced in the absence of any element, limitation, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing" and the like should be read broadly without limitation. Additionally, the terms and expressions used herein have been used as terms of description rather than limitation and are not intended to be used to exclude any equivalents or parts of features shown and described, but Various modifications can be made within the scope of the present disclosure.

Therefore, it should be understood that although the present disclosure has been specifically disclosed by specific embodiments and optional features, modifications, improvements and variations of the embodiments disclosed herein may be employed by those skilled in the art and that such modifications, improvements and variations are It is considered to be within the scope of this disclosure. The materials, methods, and examples provided herein are representative of specific embodiments, are exemplary, and are not intended to limit the scope of the present disclosure.

The scope of the disclosure has been described broadly and generically herein. Each of the narrower species and subgenus groups falling within the general disclosure also forms part of this disclosure. This includes a general description with a proviso or negative limitation removing any object from the genus, whether or not the removed material is specifically recited herein.

Additionally, where features or aspects of the present disclosure are described in terms of Markush groups, those skilled in the art will recognize that embodiments of the present disclosure may thus also be in terms of any single member or subgroup of members of the Markush group to describe.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

Claims (30)

1. a kind of side for enhancing cynapse generation in the object with cochlea cynapse disease or vestibular cynapse disease and/or neural process occurs Method, comprising applying a effective amount of 2,4- disulfonyl base α-Phenyl t-butyl Nitrone (2,4- to the object for thering is this to need ) or its pharmaceutically acceptable salt DSPBN.

2. according to the method described in claim 1, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are as medicine group Object application is closed, described pharmaceutical composition also includes pharmaceutically acceptable carrier.

3. according to the method described in claim 1, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt by oral ground, Intravenously, hypodermically, sublingually, very hypodermically, intrathecally, by sucking or in topical it is applied to the object.

4. according to the method described in claim 1, wherein the method further include application it is one or more selected from the following Compound: N-acetylcystein, acetyl-l-carnitine, glutathione mono ethyl ester, ebselen, D-Met, Carbamathione and Szeto-Schiller peptide and its functional analogue.

5. according to the method described in claim 1, wherein the method further includes application N-acetylcystein.

6. according to the method described in claim 1, wherein the object suffers from chronic acoustic trauma or chronic hearing loss.

7. according to the method described in claim 6, wherein the chronic acoustic trauma or chronic hearing loss are caused by aging 's.

8. according to the method described in claim 6, wherein the chronic acoustic trauma or chronic hearing loss are by being exposed to urgency Caused by property or chronic explosion or noise.

9. according to the method described in claim 8, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to exposure The object of at least one month after explosion or noise.

10. according to the method described in claim 8, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to cruelly It is exposed to the object of at least a year after explosion or noise.

11. according to the method described in claim 6, wherein the chronic acoustic trauma or chronic hearing loss are caused by infection 's.

12. according to the method for claim 11, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to sense The object of at least one month after dye.

13. according to the method for claim 11, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to sense The object of at least a year after dye.

14. according to the method described in claim 6, wherein the chronic acoustic trauma or chronic hearing loss are by being exposed to poison Caused by element.

15. according to the method for claim 14, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to cruelly It is exposed to the object of at least one month after toxin.

16. according to the method for claim 14, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to cruelly It is exposed to the object of at least a year after toxin.

17. according to the method described in claim 1, wherein the object also suffers from tinnitus.

18. according to the method described in claim 1, wherein the object also suffers from hyperakusis.

19. according to the method described in claim 1, wherein the object also suffers from presbycusis.

20. according to the method described in claim 1, wherein the object also suffers from disequilibrium or the plum Buddhist nun with synapse loss Angstrom disease.

21. according to the method described in claim 1, wherein the application of 2, the 4-DSPBN or its pharmaceutically acceptable salt increases The regeneration that cochlea nerve in the strong object is prominent or vestibular nerve is prominent.

22. according to the method described in claim 1, wherein on the inner hair cell in the object it is active nerve connection Number increases.

23. according to the method described in claim 1, wherein cynapse in the audio region in the corti's organ in the object Quantity increases.

24. according to the method described in claim 1, wherein the object does not undergo the reality of cochlear hair cell or vestibular hair cells Matter loss.

25. according to the method described in claim 1, wherein the object has gone through cochlear hair cell or vestibular hair cells Any substantial loss.

26. according to the method described in claim 1, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to and listen The object of at least one month after feel damage or hearing loss.

27. according to method described in claim 1, wherein 2, the 4-DSPBN or its pharmaceutically acceptable salt are applied to the sense of hearing The object of at least a year after damage or hearing loss.

28. it is a kind of for reducing the neurodegenerative method in object, comprising applying effective quantity to the object for thering is this to need 2,4- disulfonyl base α-Phenyl t-butyl Nitrone (2,4-DSPBN) or its pharmaceutically acceptable salt.

29. a kind of method for reducing the accumulation of the Tau albumen in object, comprising having to the object application for thering is this to need 2,4- disulfonyl base α-Phenyl t-butyl Nitrone (2,4-DSPBN) of effect amount or its pharmaceutically acceptable salt.

30. a kind of side for enhancing cynapse generation and neural process generation in the object with central nervous system disease or illness Method, comprising applying a effective amount of 2,4- disulfonyl base α-Phenyl t-butyl Nitrone (2,4- to the object for thering is this to need ) or its pharmaceutically acceptable salt DSPBN.